WO2022267942A1

WO2022267942A1 - Polypeptides having specific spatial structures and application thereof in ipsc preparation

Info

Publication number: WO2022267942A1
Application number: PCT/CN2022/098836
Authority: WO
Inventors: 尹乐; 顾雨春; 吴理达
Original assignee: 呈诺再生医学科技(珠海横琴新区)有限公司
Priority date: 2021-06-22
Filing date: 2022-06-15
Publication date: 2022-12-29
Also published as: CN113563428B; CN113563428A

Abstract

Polypeptides having specific spatial structures and an application thereof in preparing iPSCs are provided, amino acid sequences of the polypeptides having the specific spatial structures being as shown in SEQ ID NOs. 1-5. Also provided are nucleic acid molecules encoding the polypeptides, constructs comprising the nucleic acid molecules, and products comprising the polypeptides. By means of expressing the polypeptide sequences having the specific spatial structures, iPS can be induced and obtained, which can shorten the time required for inducing iPS cells and increase the success rate of induction.

Description

Specific Spatial Structure Peptides and Their Application in Preparation of iPSCs

technical field

The present invention relates to the field of cell engineering, in particular to specific spatial structure polypeptides and their application in preparing iPSCs.

Background technique

Stem cells are a type of cells capable of self-renewal through cell mitosis, and can differentiate into various specialized cells under specific conditions. This self-renewal capacity of stem cells underlies the cellular differentiation and specialization required for organ and tissue development. Recent evidence shows that stem cells can be used for tissue reconstruction and restoration of physiological function and tissue function. Therefore, stem cells have the potential to be used in the treatment of a variety of diseases and injuries, including nervous system trauma, malignancy, genetic disorders, hemoglobinopathies, and immune deficiencies. Stem cell transplantation therapy is an important direction of regenerative medicine research. Stem cell transplantation can be used to treat heart injury, nervous system degenerative disease, spinal cord injury, kidney failure, blood system disease and so on. However, stem cell transplantation therapy faces many difficult problems such as allogeneic rejection and limited cell sources.

Induced pluripotent stem cells (iPSC, induced pluripotent stem cells) are similar to embryonic stem cells and have developmental pluripotency. By introducing specific genes into somatic cells, they can induce somatic cell reprogramming to obtain stem cell characteristics.

In 2006, the laboratory of Yamanaka (Shinya Yamanaka), a professor at Kyoto University in Japan, announced for the first time that the introduction of four genes (Oct4, Sox2, c-Myc and Klf4) into mouse fibroblasts successfully induced mouse fibroblasts to regenerate. Programmed as totipotent stem cells, the resulting stem cells have properties similar to those of embryonic stem cells. Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) by using classical Yamanaka factors (i.e., Oct4, Sox2, Klf4, and Myc, also known as OSKM factors) has achieved great success in determining cell fate as well as in regenerative medicine. value research results. Reprogramming factors different from OSKM factors have been reported, which can induce the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells, but their induction efficiency is low and the reproducibility is low. So far, no other factor for inducing somatic cell reprogramming that can replace Yamanaka factor with the same induction efficiency or better induction efficiency than Yamanaka factor has been found.

Contents of the invention

The invention relates to a novel iPS (induced pluripotent stem cell) induction method. Somatic cells can be induced to obtain iPS (induced pluripotent stem cells) by expressing a polypeptide sequence with a specific spatial structure, shortening the time required for inducing iPS cells, and simultaneously Improve induction success rate. The iPS inducing factor involved in the present invention is based on protein structure design, which is different from the traditional method of expressing biologically derived genes (such as Oct4, Sox2, etc.), and is a brand new iPS preparation method.

The present invention provides a specific spatial structure polypeptide, the polypeptide has at least one of the following amino acid sequences:

1) The amino acid sequence shown in at least one of SEQ ID NO.1-5;

2) a part of the amino acid sequence shown in at least one of SEQ ID NO.1-5;

3) An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, further preferably at least 99% sequence identity with the amino acid sequence shown in (1 and (2).

Further, having at least 80%, preferably at least 90%, more preferably at least 95%, further preferably at least 99% sequence identity refers to having at least 80%, 81%, 82%, 83%, 84%, 85%, 86% %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

The present invention also provides a fusion protein, which comprises the above-mentioned polypeptide and other polypeptides, and the other polypeptides are connected to the C-terminus of the above-mentioned polypeptide.

Further, other polypeptides include polypeptides that promote transcriptional expression of proteins.

Further, other polypeptides are connected to the aforementioned polypeptides through GS linker.

Further, the GS linker that can be used in the present invention includes (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, wherein, n is a natural number.

Preferably, the other polypeptide is VP32; preferably, the amino acid sequence of the VP32 is shown in SEQ ID NO.11.

The present invention also provides a nucleic acid molecule comprising a first nucleic acid molecule encoding a polypeptide having at least one of the following amino acid sequences:

1) The amino acid sequence shown in at least one of SEQ ID NO.1-5;

2) a part of the amino acid sequence shown in at least one of SEQ ID NO.1-5;

Further, the first nucleic acid molecule has at least one of the following nucleotide sequences:

1) The nucleotide sequence shown in at least one of SEQ ID NO.6-10;

2) A part of the nucleotide sequence shown in at least one of SEQ ID NO.6-10;

3) A nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, further preferably at least 99% sequence identity with the nucleotide sequence shown in (1 and (2);

4) a nucleotide sequence that hybridizes to at least one of the nucleotide sequences shown in SEQ ID NO.6-10 under stringent hybridization conditions.

In a specific embodiment of the present invention, the nucleic acid molecule also includes a second nucleic acid molecule, and the second nucleic acid molecule encodes other polypeptides described above; preferably, the nucleotide sequence of the other polypeptides is as SEQ ID NO .12.

The present invention also provides a construct comprising the aforementioned nucleic acid molecule.

In a specific embodiment, the nucleotide sequences described in SEQ ID NO.6-10 can be constructed on a construct at the same time, and the four nucleotide sequences can be connected sequentially through the Linker sequence, and the sequence is not limited .

In a specific embodiment, the nucleotide sequences described in SEQ ID NO.6-10 are each individually constructed on a construct.

In a specific embodiment, any two of the nucleotide sequences described in SEQ ID NO.6-10 can be constructed on one construct. Different nucleotide sequences are connected sequentially through the Linker sequence, and the order of arrangement is not limited. The other two nucleotide sequences can be constructed on one construct, or each nucleotide sequence can be constructed on one construct separately.

In a specific embodiment, any three of the nucleotide sequences described in SEQ ID NO.6-10 can be constructed on one construct. Different nucleotide sequences are connected sequentially through the Linker sequence, and the order of arrangement is not limited. An additional nucleotide sequence is constructed separately on a construct.

The Linker sequence may be a coding sequence of a self-cleaving peptide, and the self-cleaving peptide includes P2A, T2A, F2A, E2A, BmCPV2A, and BmIFV2A.

Further, the construct also includes regulatory elements operably linked to the aforementioned nucleic acid molecules.

A "regulatory element" of the present invention is an element necessary for translation and/or transcription of an inserted coding sequence. are those untranslated regions (enhancer, promoter, 5' and 3' untranslated regions) of the constructs described below that interact with host cell proteins for transcription and translation. Such elements may vary in strength and specificity.

Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue-specific or developmental stage-specific promoters, inducible promoters, and synthetic promoters.

Constitutive promoters direct expression in virtually all tissues and are largely independent of environmental and developmental factors. Because their expression is generally not regulated by endogenous factors, constitutive promoters are often active across species and even kingdoms. Examples of constitutive promoters include CMV, EF1a, SV40, PGK1, Ubc, human beta actin, and CAG.

In a particular embodiment of the invention, said promoter is the EF1α promoter.

Preferably, the construct includes one or more of plasmids, phages, artificial chromosomes, cosmids, and viruses.

Preferably, the construct is a plasmid.

Methods for constructing constructs containing genetic sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Press, Plainview, N.Y., 1989), and Ausubel et al., Current Protocols in Molecular Biology [Modern Methods in Molecular Biology] (John Wiley & Sons [John Wiley & Sons], New York City, NY, 1989).

For example, a recombinant vector is constructed by inserting the target gene sequence into a plasmid vector. Examples of plasmid vectors include, but are not limited to, pEF1α plasmid vector, pBR322 plasmid vector, pUC plasmid vector, pMOB45 plasmid vector, pACYC plasmid vector, pSC101 plasmid vector, colE1 plasmid vector, and the like. The method of inserting a sequence into a conventional vector to construct a recombinant plasmid vector is well known to those skilled in the art.

The present invention also provides a product for inducing somatic cells to produce induced pluripotent stem cells, the product comprising at least one of the following:

1) the aforementioned polypeptide;

2) the aforementioned fusion protein;

3) the aforementioned nucleic acid molecules;

4) Constructs as described above.

Further, the products include compositions and kits. The composition or the kit further includes a pharmaceutically acceptable carrier in addition to the above components.

Further, the composition may also include other reprogramming factors, fusion proteins constructed of other reprogramming factors, nucleic acid molecules encoding other reprogramming factors, and constructs expressing other reprogramming factors.

Other reprogramming factors include the reprogramming factors already disclosed in the prior art, examples of other reprogramming factors are but not limited to: Oct 3/4, Sox2, Sox1, Sox3, Sox15, Sox18, Klf1, Klf2, Klf4, Klf5, c -myc, L-myc, N-myc, NANOG, LIN28, RARg, Lrh-1, P53, ECAT1, UTF1, ESRRB, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1.

Further, the composition can also be a culture medium.

"Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject with a protein, nucleic acid, or carrier without causing any undesired biological effects .

Pharmaceutically acceptable carriers are known to those skilled in the art. These will most typically be the standard carriers used to administer drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Compositions can be administered intramuscularly or subcutaneously.

Specifically, the composition may include carriers, thickeners, diluents, buffers, preservatives, surfactants and the like. The compositions may also include one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, etc. may also be present.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavourings, diluents, emulsifiers, dispersing aids or binders may be desired.

The compositions disclosed herein, including pharmaceutical compositions, can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. It can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, in vitro, ophthalmically, vaginally, rectally, intranasally, topically, etc. (including topical intranasal administration or by inhalation agent application) composition.

The present invention also provides an engineered cell or population thereof, and the engineered cell or population thereof expresses the aforementioned polypeptide or the aforementioned fusion protein.

Further, the engineered cells or their populations are intermediate cells between somatic cells and induced pluripotent stem cells, or called reprogrammed cells. Induced pluripotent stem cells are cells that have been reprogrammed. A reprogrammed cell is a cell that is at a lower state of differentiation than the same cell in a non-reprogrammed state. In contrast to cells that have been reprogrammed, "reprogrammed cells" refer to non-pluripotent cells that are undergoing reprogramming/dedifferentiation to a pluripotent state, exhibiting a transitional morphology (i.e., morphological change), but without hallmarks of pluripotent cells , including pluripotent stem cell morphology or stable endogenous pluripotency gene expression, such as OCT4, NANOG, SOX2, SSEA4, TRA181, CD30 and/or CD50. The transitional morphology of the reprogrammed cells distinguishes the cells from the initial non-pluripotent cells prior to reprogramming induction and from reprogrammed cells with embryonic stem cell hallmark morphology. Such transitional morphologies of induced reprogrammed various types of somatic cells are readily understood and identified by those skilled in the art.

In particular embodiments of the invention, the reprogrammed cells are those that have been induced to reprogram for at least 1, 2, 3, 4, 5, 6, 7, 8 or more days, but not exceeding any number of days from 16 to 22 days Intermediate cells, wherein the cells have not yet entered a self-sustaining or self-sustaining pluripotent state. Unlike somatic cells prior to exposure to exogenously expressed reprogramming factors, reprogrammed cells can progress the reprogramming process to a stable pluripotent state, even in the absence of exogenously expressed reprogramming factors, given sufficient time. into reprogrammed cells.

In some embodiments, the engineered cell or population thereof comprises the aforementioned nucleic acid molecule or the aforementioned construct.

In some embodiments, the engineered cell or population thereof does not comprise the aforementioned nucleic acid molecule or the aforementioned construct.

The present invention also provides a method for preparing the above-mentioned engineered cell or its population, the method comprising introducing the above-mentioned polypeptide, the above-mentioned fusion protein, and the above-mentioned nucleic acid molecule into the cell , one or more of the aforementioned constructs.

Cells for introduction can be of any type, including but not limited to somatic cells.

The present invention also provides a method for inducing somatic cells to produce induced pluripotent stem cells, the method comprising introducing the aforementioned polypeptide, the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned One or more of the constructs.

Further, the method includes the following steps:

1) introducing one or more of the aforementioned polypeptides, the aforementioned fusion proteins, the aforementioned nucleic acid molecules, and the aforementioned constructs into somatic cells;

2) Inducing and culturing the somatic cells treated in step 1) to obtain induced pluripotent stem cells.

Somatic cells suitable for use in the present invention may be stem cells or mature cells. These cells may be referred to as "donor cells". Adult stem cells are undifferentiated cells found throughout the body. They can proliferate through cell division to replenish dead cells and regenerate damaged tissue. Adult or somatic stem cells have been identified in many organs and tissues including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, intestine, liver, ovarian epithelium and testis. They are thought to reside in specific areas of each tissue known as "stem cell nests" and provide a source of cells for that tissue only. Types of adult stem cells include hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cells.

If mature cells are harvested as donor cells, they may be from any tissue, organ, body fluid or body secretion. Thus, the cells may be skin cells, hair follicle cells, blood cells, cells extracted from urine, cells collected by biopsy or the like from any tissue or organ including, but not limited to, bones, teeth, dental tissue, Heart, lungs, brain, pancreas, liver, kidneys, bladder, uterus, intestines, stomach, gallbladder, muscle, fat, testes, mucous membranes, eyes, foreskin, prostate, spleen or any other tissue.

In a particular embodiment of the invention, said somatic cells are peripheral blood mononuclear cells;

The somatic cells of the present invention are derived from mammals. The mammal includes human, rat, mouse, monkey, dog, cat, cow, rabbit, horse, pig.

Preferably, the somatic cells are of human origin.

In the method of the present invention, the aforementioned polypeptide or fusion protein can be expressed in vitro, and then introduced into cells to achieve the purpose of the present invention. Methods for introducing polypeptides or fusion proteins into cells are well known in the art, including but not limited to Tat-delivery and related technologies, electroporation (nucleofection), gene gun, SLO cell permeabilization, and binding of proteins and cell ligands.

In the method of the present invention, the mRNA or cDNA of the above-mentioned polypeptide or fusion protein can be directly introduced into the cell, so that it can be expressed in the cell to produce the corresponding protein, so as to achieve the purpose of the present invention. Techniques for introducing mRNA or cDNA into cells are similar to techniques for introducing proteins into cells and are well known in the art.

In the method of the present invention, the mRNA or cDNA of the aforementioned polypeptide or fusion protein can be loaded into a vector to form a construct, and then the construct can be introduced into a cell to express the corresponding protein in the cell, thereby realizing purpose of the present invention. Ways to introduce constructs into cells are well known in the art, including but not limited to electroporation, chemical transfection.

Furthermore, in a specific embodiment of the present invention, the specific operation process of the above-mentioned method is:

a) electrotransfecting the plasmid vector expressing the polypeptide shown in SEQ ID NO.1-5 into peripheral blood mononuclear cells;

b) The culture medium was replaced after 24 hours of electroporation, and the culture medium was replaced after 48 hours;

c) Culture the cells on the third day after electroporation in the reprogramming medium, and replace the medium once every two days;

Preferably, the reprogramming medium formula is as shown in Table 1:

Table 1 Reprogramming Medium Recipe

d) Culture the cells on the 17th day after electroporation in the stem cell medium, and update the medium every day;

Preferably, the stem cell medium is TeSR ^™ -E8 ^™ medium (Stemcell #05991, #05992).

e) The cells on day 25-28 after electroporation (when the IPSc cells form obvious and large clones) are subjected to the following operations: pick out iPSCs clones for expansion and culture.

Preferably, the specific operation of step a) is: count the peripheral blood mononuclear cells obtained from blood separation, and resuspend them with electrotransfer fluid, 105 μl electrotransfer fluid per needle (100 μl volume per needle), at a mass ratio of 1:1 A total of 1 μg of each of the five plasmids was added, for a total of 5 μg of plasmids.

Preferably, 10 ⁶ cells are electroporated per needle.

Preferably, the electrotransfer parameters are 1400-1650V, 10-30ms, 1-3plus.

Preferably, the method of electroporation culture is as follows: cells after electroporation are placed in a culture dish incubated with matrigel, and cultured in a 37° C., 5% CO ₂ incubator.

The present invention also provides an induced pluripotent stem cell or its population prepared by the aforementioned method.

The present invention also provides the use of induced pluripotent stem cells or populations thereof. They can be further differentiated to produce different cell types, for example, neural stem cells and their precursors, hematopoietic stem cells and their precursors, cardiomyocytes and their precursors, endothelial progenitors and their precursors, endothelial cells and their precursors Somatic cells, islet cells and their precursors, immune cells and their precursors, retinal epithelial cells and their precursors. The induced cells and cells differentiated therefrom can be used in cell transplantation therapy. Because induced cells can be derived from non-embryonic cells, cell transplantation therapy involves providing the subject with cells derived from his or her own tissue, thereby reducing the potential for rejection.

The induced pluripotent stem cells prepared according to the method of the present invention or cells differentiated from their populations can be used for cell transplantation therapy to treat nerve cell diseases, such as stroke, Parkinson's, senile dementia, gradual frostbite, spinal cord injury, and craniocerebral injury; Blood system diseases, such as myeloma, lymphoma, neutropenia, leukemia, aplastic anemia; cardiac system diseases, such as heart failure; cardiovascular diseases, such as endothelial disorder syndrome, atherosclerosis, hypertension complications, Heart failure, acute myocardial infarction, diabetes complications; type I diabetes; various cancers, such as cervical cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, leukemia; visual diseases, such as age-related macular degeneration.

Induced pluripotent stem cells or their populations can also be used for the screening of chemotherapy drugs, and the iPSCs prepared by the method of the present invention are further differentiated into cells of specific cell lines, and then the drugs to be screened act on various types of cells , to determine the cytotoxicity and effectiveness of the drug to be screened, etc., so as to screen out the drug with the lowest cytotoxicity and the highest efficacy.

The present invention also provides applications in the following other aspects, which include any of the following:

1) The application of a specific spatial structure polypeptide or a fusion protein containing it in inducing somatic cells to produce induced pluripotent stem cells;

2) The application of the fusion protein containing the specific spatial structure polypeptide in the preparation of products for inducing somatic cells to produce induced pluripotent stem cells;

3) The application of the fusion protein containing the specific spatial structure polypeptide in the preparation of engineered cells or their populations;

4) The application of the fusion protein containing the specific spatial structure polypeptide in the production of products that produce engineered cells or their populations;

5) the application of the aforementioned nucleic acid molecules in inducing somatic cells to produce induced pluripotent stem cells;

6) Application of the aforementioned nucleic acid molecules in the preparation of products for inducing somatic cells to produce induced pluripotent stem cells;

7) Application of the aforementioned nucleic acid molecules in the preparation of engineered cells or populations thereof;

8) Application of the aforementioned nucleic acid molecules in the preparation of products producing engineered cells or populations thereof;

9) application of the aforementioned nucleic acid molecules in the preparation of the aforementioned constructs;

10) Application of the aforementioned constructs in inducing somatic cells to produce induced pluripotent stem cells;

11) Application of the aforementioned construct in the preparation of products for inducing somatic cells to produce induced pluripotent stem cells; preferably, the product is the aforementioned product;

12) Use of the aforementioned constructs in the preparation of engineered cells or populations thereof;

13) Use of the aforementioned constructs in the production of products producing engineered cells or populations thereof;

14) Application of the aforementioned engineered cells or populations thereof in the preparation of induced pluripotent stem cells;

15) Application of the above-mentioned engineered cells or their populations in the preparation of products promoting the production of induced pluripotent stem cells.

Preferably, said somatic cells are as defined above.

Preferably, the specific spatial structure polypeptide or the fusion protein comprising it is the aforementioned polypeptide or the aforementioned fusion polypeptide.

Preferably, said product is a product as previously described.

The term "stringent conditions" as used herein refers to conditions under which specific hybrids are formed and non-specific hybrids are not formed. Typical stringent conditions include, for example, conditions in which hybridization is performed at a potassium concentration of about 25 mmol/L to about 50 mmol/L and a magnesium concentration of about 1.0 mmol/L to about 5.0 mmol/L. Examples of the conditions of the present invention include, but not limited to, conditions for hybridization under Tris-HCl (pH 8.6), 25 mmol/L KCl, and 1.5 mmol/L MgCl ₂ . In addition, stringent conditions are described in Molecular Cloning 3rd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001). This document is incorporated into this specification by reference. Those skilled in the art can easily select the above-mentioned conditions by changing the hybridization reaction, the salt concentration of the hybridization reaction solution, and the like.

The term "identity" used herein is well known in the art and refers to the relationship between two or more nucleic acid sequences determined by sequence comparison; in the art, "identity" also refers to the relationship between nucleic acid molecules The degree of sequence relatedness between sequences is determined by the degree of base matching of those sequences; "identity" can be readily calculated according to known methods including, but not limited to, those described in Computer Molecular Biology, Lesk, ed. Oxford University Press, New York, 1993; Biocomputing: Infornatics and Genome Projects, edited by Smith, Academic Publishing, New York, 1988; Computer Analysis of Sequence Data, Part I, edited by Griffin and Griffin, Humana Publishing, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, Academic Publishing 1987; Sequence Analysis Primer, edited by Gribskov and Devereux, Stockton Publishing, New York 1991; and Carillo and Lipman, SIAM J, Applied Mathematics, 48:1073, 1988. ).

The term "polypeptide" refers to amino acids linked to each other by peptide bonds or modified peptide bonds (eg, peptide isosteres, etc.), and may contain modified amino acids other than the 20 gene-encoded amino acids. Polypeptides can be modified by natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. The same type of modification can exist to the same or different degrees at several positions in a given polypeptide. Likewise, a given polypeptide may have many types of modifications. Modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent crosslinking or cyclization, covalent attachment of flavin, covalent attachment of heme moieties, nucleotides or nucleotides Covalent attachment of derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositols, disulfide bond formation, demethylation, cysteine or pyroglutamate formation , formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, isopentenyl ylation, racemization, selenylation, sulfation, and transfer RNA-mediated addition of amino acids to proteins (eg, arginylation) (see Proteins-Structure and Molecular Properties, 2nd ed., T.E. Creighton, W.H. Freeman and Company [W.H. Freeman Company], New York (1993); Posttranslational Covalent Modification of Proteins [post-translational covalent modification of proteins], edited by B.C. Johnson, Academic Press [Academic Press], New York, No. 1- 12 pages (1983)).

Description of drawings

Figure 1 shows the cell morphology on the 6th day after electroporation, where A: control group; B: experimental group;

Figure 2 shows the cell morphology on the 9th day after electroporation, where A: control group; B: experimental group;

Figure 3 shows the cell morphology on the 13th day after electroporation, where A: control group; B: experimental group;

Figure 4 shows the cell morphology on the 17th day after electroporation, where A: control group; B: experimental group;

Figure 5 shows the results of detecting stem cell markers expressed by cells in the control group by immunofluorescence imaging experiments;

Figure 6 shows the results of detecting stem cell markers expressed by cells in the experimental group by immunofluorescence imaging experiments;

Fig. 7 shows a schematic diagram of the structure of the recombinant plasmid vector constructed in the present invention.

detailed description

The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific techniques or conditions are indicated in the examples, according to the techniques or conditions described in the literature in this field (for example, refer to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang, third edition, Science Press) or follow the product instructions. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Embodiment 1 Polypeptide expression vector construction

1. Steps

The nucleic acid sequence encoding the polypeptide shown in Synthesis SEQ ID NO.1-5 is shown in SEQ ID NO.6-10, each nucleic acid sequence is inserted into the pEF1α vector respectively, and the schematic diagram of the recombinant expression vector constructed is shown in Figure 7 (pep1-pep5 refer to polypeptides). Each vector contains a main open reading frame (ORF), expresses a polypeptide from the EF1α promoter, and fuses VP32 protein at the C-terminus. VP32 is connected to pep through a GS linker (such as GGGS), which can improve the efficiency of transcription and expression.

Example 2 iPSC induction

Taking peripheral blood mononuclear cells as an example, the steps of using the polypeptide of the present invention to induce iPSC production are described. Control group and experiment group are set, and control group is wild-type Sox2, Oct4, Klf4, C-myc (amino acid sequence and nucleotide sequence can be inquired on NCBI) expression plasmid (construction method is the same as embodiment 1); Experimental group It is the plasmid vector constructed in Example 1 of the present invention.

1. Steps

Day 1:

1. Count the peripheral blood mononuclear cells obtained from blood separation, and resuspend them with electroporation fluid. Add 1 μg of each of the five plasmids to 105 μl of electroporation fluid per needle (100 μl volume per needle) at a mass ratio of 1:1, for a total of 5 μg of plasmids. , by electroporation of 10 ⁶ cells per needle;

2. The parameters of the electric transfer are 1400~1650V, 10~30ms, 1~3plus;

3. Spread the electrotransferred cells into the prepared culture dish (the culture dish needs to be incubated with matrigel);

4. Culture the cells in 3 in a 37°C, 5% CO ₂ incubator;

5. Replace the medium after culturing the cells in 4 for 24 hours, and replace the medium after 48 hours.

Day 3 to 20:

The cells were cultured in the reprogramming medium (shown in Table 1), and the medium was changed every two days.

Table 1 Reprogramming Medium Recipe

Day 20-28:

Cells were cultured in stem cell medium TeSR ^TM -E8 ^TM (stemcell Catalog #05990), and the medium was renewed every day.

Day 28:

Pick out iPSCs clones to 96 or 48 or 24-well plates (depending on the clone size and growth length) for expansion culture, and finally scale up to T175 culture flasks (depending on the end use).

2. Results

As shown in Figure 1, on the 6th day after electroporation, the experimental group began to have obvious deformed cell attachment (IPS precursor cells), and had begun to proliferate into clones, while only a small number of cells in the control group began to adhere to the wall.

As shown in Figure 2, on the 9th day after electroporation, obvious cell clones could be observed in the experimental group, and at this time clones in the control group began to form.

As shown in Figure 3, on the 13th day after electroporation, the clones in the experimental group became significantly larger, and the cell expansion was obvious. The control group has not yet formed obvious clones, and the number of cells decreased compared with the ninth day, indicating that there is apoptosis and the formation of clones is unstable.

As shown in Figure 4, on the 17th day after electroporation, obvious iPS clones had formed in the experimental group and could be picked and cultured, while no obvious single clones had been formed in the control group and continued to be cultured.

The above results show that after electroporation of the plasmid vector expressing the polypeptide sequence of the present invention, peripheral blood mononuclear cells (suspension cells) can be induced to become iPS cells (adherent cells) after about 17 days, while conventional methods (electroporation expressing Sox2, Oct4, Klf4, C-myc plasmids) generally need more than 20 days to obtain iPS single clones. While shortening the iPS acquisition time, the method of the present invention also improves the efficiency of inducing iPS. Statistics show that iPS single clones in 3 six-well plates have 18 holes in total. The number of clones, the iPS induction method involved in the present invention is more than 5 times that of the traditional method (n=3, p<0.025).

3. Detection of characteristic markers of stem cells

The expressions of related markers in iPSCs prepared from the control group and the experimental group were detected by staining immunofluorescence imaging experiments. The results are shown in Figure 5 and Figure 6, both the control group and the experimental group expressed endogenous stem cell markers Nanog, Oct4, SSEA-4, TRA-1-60. The results show that the method of the present invention can successfully prepare iPSCs.

Claims

A specific spatial structure polypeptide, characterized in that the polypeptide has at least one of the following amino acid sequences:

1) The amino acid sequence shown in at least one of SEQ ID NO.1-5;

2) a part of the amino acid sequence shown in at least one of SEQ ID NO.1-5;

3) An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, further preferably at least 99% sequence identity with the amino acid sequence shown in (1 and (2).
A fusion protein, characterized in that, the fusion protein comprises the polypeptide according to claim 1 and other polypeptides, and the other polypeptides are connected to the C-terminus of the polypeptide according to claim 1; preferably, the other polypeptides and The polypeptides are linked by a GS linker; preferably, the GS linker includes (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, wherein, n is a natural number; preferably, the other polypeptides include promoting proteins The polypeptide expressed by transcription; preferably, the other polypeptide is VP32; preferably, the amino acid sequence of the VP32 is shown in SEQ ID NO.11.
A nucleic acid molecule, characterized in that, the nucleic acid molecule comprises a first nucleic acid molecule, and the first nucleic acid molecule has at least one of the following nucleotide sequences:

1) The nucleotide sequence shown in at least one of SEQ ID NO.1-5;

2) A part of the nucleotide sequence shown in at least one of SEQ ID NO.1-5;

3) A nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, further preferably at least 99% sequence identity with the nucleotide sequence shown in (1 and (2).

Preferably, the first nucleic acid molecule has at least one of the following nucleotide sequences:

1) The nucleotide sequence shown in at least one of SEQ ID NO.6-10;

2) A part of the nucleotide sequence shown in at least one of SEQ ID NO.6-10;

3) A nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, further preferably at least 99% sequence identity with the nucleotide sequence shown in (1 and (2);

4) a nucleotide sequence that hybridizes to at least one of the nucleotide sequences shown in SEQ ID NO.6-10 under stringent hybridization conditions;

Preferably, the nucleic acid molecule also comprises a second nucleic acid molecule, the second nucleic acid molecule encodes other polypeptides described in claim 2; preferably, the nucleotide sequence of the other polypeptides is as shown in SEQ ID NO.12 Show.
A construct, characterized in that, the construct comprises the nucleic acid molecule of claim 3; preferably, the construct further comprises a regulatory element operably linked to the nucleic acid molecule of claim 3; preferably Preferably, the regulatory elements include a promoter, an enhancer; preferably, the promoter is an EF1α promoter; preferably, the construct includes one or more of a plasmid, a phage, an artificial chromosome, a cosmid, and a virus species; preferably, the construct is a plasmid; preferably, the plasmid is a pEF1α plasmid.
A product for inducing somatic cells to produce induced pluripotent stem cells, characterized in that the product includes at least one of the following:

1) The polypeptide according to claim 1;

2) the fusion protein according to claim 2;

3) the nucleic acid molecule of claim 2;

4) the construct according to claim 3;

Preferably, the product includes a composition, a kit;

Preferably, the composition or the kit further includes a pharmaceutically acceptable carrier;

Preferably, the composition also includes other reprogramming factors, fusion proteins constructed by other reprogramming factors, nucleic acid molecules encoding other reprogramming factors, and constructs expressing other reprogramming factors;

Preferably, other reprogramming factors include reprogramming factors already disclosed in the prior art, examples of other reprogramming factors but not limited to: Oct 3/4, Sox2, Sox1, Sox3, Sox15, Sox18, Klf1, Klf2, Klf4, Klf5, c-myc, L-myc, N-myc, NANOG, LIN28, RARg, Lrh-1, P53, ECAT1, UTF1, ESRRB, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1.
An engineered cell or population thereof, characterized in that the engineered cell or population thereof expresses the polypeptide of claim 1 or the fusion protein of claim 2;

Preferably, the engineered cell or population thereof comprises the nucleic acid molecule of claim 3 or the construct of claim 4;

Preferably, the engineered cell or population thereof does not comprise the nucleic acid molecule of claim 3 or the construct of claim 4 .
A method for preparing the engineered cell or population thereof according to claim 6, characterized in that the method comprises introducing the polypeptide according to claim 1, the fusion protein according to claim 2, the fusion protein according to claim 2, or the One or more of the nucleic acid molecule of claim 3, the construct of claim 4.
A method for inducing somatic cells to produce induced pluripotent stem cells, characterized in that the method comprises introducing the polypeptide according to claim 1, the fusion protein according to claim 2, and the nucleic acid according to claim 3 into somatic cells One or more in molecule, the construct described in claim 4; Preferably, described method comprises the following steps:

1) introducing one or more of the polypeptide according to claim 1, the fusion protein according to claim 2, the nucleic acid molecule according to claim 3, and the construct according to claim 4 into somatic cells;

2) Inducing and culturing the somatic cells treated in step 1) to obtain induced pluripotent stem cells;

Preferably, said somatic cells comprise stem cells or mature cells;

Preferably, the somatic cells are peripheral blood PBMCs;

Preferably, the somatic cells are derived from mammals; preferably, the somatic cells are derived from humans;

Preferably, the introduction method includes gene gun, electroporation, Tat-delivery and related technologies, SLO cell permeation, protein and cell ligand binding, chemical transfection;

Preferably, the treatment time of step 2) is at least 16 days;

Preferably, the induction culture of the somatic cells treated in step 1) uses a reprogramming medium;

Preferably, the medium is replaced 24h after introduction in step 1), and the medium is replaced after 48h;

Preferably, when the somatic cells are peripheral blood mononuclear cells, the mass ratio of the electroporation resuspension of peripheral blood mononuclear cells to the plasmid is 1:1;

Preferably, 10 6 cells are electroporated per needle.

Preferably, the electrotransfer parameters are 1400-1650V, 10-30ms, 1-3plus.

Preferably, the method of electroporation culture is as follows: cells after electroporation are placed in a culture dish incubated with matrigel, and cultured in a 37° C., 5% CO 2 incubator.
Induced pluripotent stem cells or populations thereof prepared by the method of claim 8.
An application, characterized in that the application includes any of the following:

1) The application of a specific spatial structure polypeptide or a fusion protein containing it in inducing somatic cells to generate induced pluripotent stem cells;

2) The application of the fusion protein containing the specific spatial structure polypeptide in the preparation of products for inducing somatic cells to produce induced pluripotent stem cells;

3) Application of a fusion protein comprising a specific spatial structure polypeptide in preparing engineered cells or populations thereof; preferably, the engineered cells or populations thereof are the engineered cells according to claim 6 Modified cells or populations thereof;

4) The application of a fusion protein comprising a polypeptide with a specific spatial structure in the production of products that produce engineered cells or populations thereof; preferably, the engineered cells or populations thereof are as described in claim 6 engineered cells or populations thereof;

5) the application of the nucleic acid molecule described in claim 3 in inducing somatic cells to produce induced pluripotent stem cells;

6) The application of the nucleic acid molecule according to claim 3 in the preparation of products for inducing somatic cells to produce induced pluripotent stem cells;

7) Use of the nucleic acid molecule according to claim 3 in the preparation of engineered cells or populations thereof; preferably, the engineered cells or populations thereof are the engineered cells according to claim 6 cells or populations thereof;

8) Application of the nucleic acid molecule of claim 3 in the production of products producing engineered cells or populations thereof; preferably, the engineered cells or populations thereof are the engineered cells or populations thereof as claimed in claim 6 engineered cells or populations thereof;

9) the application of the nucleic acid molecule described in claim 3 in preparing the construct described in claim 4;

10) the application of the construct described in claim 4 in inducing somatic cells to produce induced pluripotent stem cells;

11) Application of the construct according to claim 4 in the preparation of products for inducing somatic cells to produce induced pluripotent stem cells;

12) Use of the construct of claim 4 in the preparation of engineered cells or populations thereof; preferably, the engineered cells or populations thereof are engineered cells or populations thereof as claimed in claim 6 cells or populations thereof;

13) Use of the construct according to claim 4 in the production of products producing engineered cells or populations thereof; preferably, said engineered cells or populations thereof are engineered cells or populations thereof as claimed in claim 6 engineered cells or populations thereof;

14) Application of the engineered cells or populations thereof according to claim 6 in the preparation of induced pluripotent stem cells;

15) Application of the engineered cells or populations thereof according to claim 6 in the preparation of products promoting the production of induced pluripotent stem cells;

16) the application of the induced pluripotent stem cells or populations thereof according to claim 9 in cell differentiation;

17) Application of the induced pluripotent stem cells or their populations according to claim 9 in the preparation of differentiated cell products;

18) Application of the induced pluripotent stem cells or their populations according to claim 9 in the preparation of medicines for cell transplantation therapy;

19) Application of the induced pluripotent stem cells or populations thereof according to claim 9 in the screening of chemotherapeutic drugs;

20) Application of the induced pluripotent stem cells or their populations according to claim 9 in the preparation of cell lines for screening chemotherapeutic drugs;

Preferably, said somatic cells comprise stem cells or mature cells;

Preferably, the somatic cells are derived from mammals; preferably, the somatic cells are derived from humans;

Preferably, the somatic cells are peripheral blood PBMCs;

Preferably, the specific spatial structure polypeptide or the fusion protein comprising it is the polypeptide according to claim 1 or the fusion polypeptide according to claim 2;

Preferably, the product is the product of claim 5;

Preferably, the differentiated cells include neural stem cells and their precursor cells, hematopoietic stem cells and their precursor cells, cardiomyocytes and their precursor cells, endothelial progenitor cells and their precursor cells, endothelial cells and their precursor cells, pancreatic islets cells and their precursors, immune cells and their precursors, retinal epithelial cells and their precursors;

Preferably, the types of diseases treated by cell transplantation therapy include nerve cell diseases, such as stroke, Parkinson's, Alzheimer's disease, frostbite, spinal cord injury, craniocerebral injury; blood system diseases, such as myeloma, lymphoma, neutropenia disease, leukemia, aplastic anemia; cardiac system disease, such as heart failure; cardiovascular disease, such as endothelial disorder syndrome, atherosclerosis, hypertension complications, heart failure, acute myocardial infarction, diabetes complications; type I diabetes Various cancers, such as cervical cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, leukemia; visual diseases, such as age-related macular degeneration.