WO2021107234A1 - Customized ccr5/cxcr4-gene simultaneous knockout hematopoietic stem cells for treatment or prevention of hiv infection, and preparation method therefor - Google Patents

Abstract

The present invention relates to a method for preparing customized CCR5/CXCR4-gene simultaneous knockout hematopoietic stem cells by using a CRISPR/Cas9 system, and a pharmaceutical composition and a cell therapeutic agent for treating or preventing HIV infection, comprising the hematopoietic stem cells. The method for preparing a hematopoietic stem cell, of the present invention, can efficiently prepare hematopoietic stem cells in which CCR5 and CXCR4 genes are simultaneously knocked out, and thus hematopoietic stem cells prepared thereby can be used as a pharmaceutical composition and cell therapeutic agent for treating or preventing HIV infection.

Description

Simultaneous knockout of CCR5/CXCR4 gene for treatment or prevention of HIV infection, patient-specific hematopoietic stem cells and manufacturing method thereof

The present invention relates to a method for producing a patient-specific hematopoietic stem cell for simultaneous knockout of the CCR5/CXCR4 gene using the CRISPR/Cas9 system, a pharmaceutical composition for treating or preventing HIV virus infection containing the hematopoietic stem cell, and a cell therapy agent.

Human Immunodeficiency Virus (HIV) is a virus that causes Acquired Immune Deficiency Syndrome (AIDS), an acquired immunodeficiency syndrome. HIV infects and destroys helper T cells (CD4 T cells), and the infection Repeatedly, the number of helper T cells in the patient's body is extremely reduced, which eventually leads to the loss of the patient's immune function, leading to the death of HIV-infected patients from opportunistic infections or cancer.

Since the first case of AIDS was discovered in the United States in 1981, the number of infections has exploded every year, and it is estimated that there are currently about 35 million people worldwide. It is estimated that about 10,000 people are infected.

HIV is a single-stranded RNA virus that has reverse transcriptase and uses the surface gp120 protein to co-receptor with CD4 receptor on the surface of helper T cells, CC chemokine receptor type 5 (CCR5; CD195) or CXC chemokine receptor type 4 ( Infects helper T cells through a mechanism that interacts with CXCR4; CD184).

In addition, HIV is divided into two types according to the co-receptor used when infecting helper T cells. HIV using CCR5 is called R5 type, and HIV using CXCR4 is called X4 type.

Currently, for drug treatment of HIV infection, cocktail therapy using a drug that inhibits the reverse transcriptase present in HIV is mainly used. However, the mutation of HIV is so severe that it is impossible to cure it with drug therapy, and the number of HIV-infected patients is increasing every year (2.1 million people around the world every year), and the number of people dying even though they are taking antiviral drugs (1.1 million people around the world every year) is getting lower and lower. Due to the high age of HIV infection (average age of 37) and high medical expenses (about 20 million won per person per year), a method that can fundamentally treat AIDS is essential.

To date, there has been one reported case of cure for HIV infection. This cured patient was named 'Berlin patient', meaning that he was treated in Berlin. This patient had additional leukemia after infection with HIV, and it was reported that he received another person's hematopoietic stem cells (HSC) to treat leukemia, and it was reported that he was cured from HIV infection through this hematopoietic stem cell transplantation process.

A retrospective study showed that the hematopoietic stem cells transplanted from the 'Berlin patient' were innately mutated in CCR5, a co-receptor required for HIV to infect helper T cells, so that the CCR5 mutated hematopoietic stem cells were HIV-resistant. By making helper T cells, it was confirmed that AIDS was cured.

However, there have been reports of cases where hematopoietic stem cells with mutated CCR5 were transplanted, but died without being cured of AIDS. However, it seems that this patient died without being cured because he was infected with both R5 and X4 types of HIV.

On the other hand, the CRISPR/Cas9 system is the most recently developed third-generation gene scissors and is a technology that is being actively used worldwide to knock out cellular genes. The CRISPR/Cas9 system consists of a guide RNA (gRNA) and a Cas9 nuclease, and when the gRNA introduced into the cell binds to the site to be knocked out on the genomic DNA, the Cas9 nuclease recognizes this and cuts the genomic DNA site. gene can be knocked out. Therefore, using the CRISPR/Cas9 system seems to be able to simultaneously knock out CCR5 and CXCR4 in the patient's somatic cells.

Under this background, the present inventors have developed an expression vector capable of simultaneously knocking out CCR5 and CXCR4 genes using the CRISPR/Cas9 system and a composition comprising the same, and using this, CCR5 and CXCR4 genes are knocked out hematopoietic stem cells. It was confirmed that it can be manufactured. In addition, the present invention was completed by confirming that the engraftment ability of the differentiated cells was improved by using the serum-free / xeno-free / gene-free method in the differentiation stage and culturing the stem cells by adding an antioxidant to the medium.

One object of the present invention is to (a) reducing the expression level of CCR5 and CXCR4 in an isolated cell compared to the intrinsic expression level using a gene editing technique; and (b) culturing the isolated cells of step (a) in serum-free, xeno-free and gene-free methods to differentiate them into hematopoietic stem cells. It is to provide a method for producing hematopoietic stem cells comprising a.

Another object of the present invention is to differentiate into hematopoietic stem cells through direct reprogramming from (a) isolated somatic cells; and (b) reducing the CCR5 and CXCR4 expression levels of the hematopoietic stem cells of step (a) compared to the intrinsic expression level, to provide a method for producing hematopoietic stem cells.

Another object of the present invention is to provide hematopoietic stem cells, prepared by the above method.

Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of HIV virus infection, comprising the hematopoietic stem cells as an active ingredient.

Another object of the present invention is to provide a cell therapy agent comprising the hematopoietic stem cells as an active ingredient.

Another object of the present invention is to improve the engraftment ability of stem cells by culturing stem cells in serum-free, xeno-free and gene-free methods. to provide a way

Another object of the present invention is to provide a method for improving the engraftment ability of stem cells by culturing the stem cells in a medium containing an antioxidant.

The method for producing hematopoietic stem cells of the present invention can efficiently produce hematopoietic stem cells in which the CCR5 and CXCR4 genes are simultaneously knocked out, and the hematopoietic stem cells prepared therefrom can be used as pharmaceutical compositions and cell therapies for the treatment or prevention of HIV virus infection. can

1 is a diagram schematically illustrating a guide RNA design process targeting CCR5 and CXCR4 genes.

Figure 2 is a diagram showing the experimental results of establishing electroporation conditions for the introduction of the CRISRPR / Cas9 vector.

3 is a view confirming that the CCR5 and CXCR4 genes are knocked out using the CRISRPR/Cas9 system of the present invention.

4 is a view confirming that monoallelic and biallelic knockout of the CXCR4 gene using the CRISRPR/Cas9 system of the present invention.

5 is a view confirming the production of induced pluripotent stem cells (iPSCs);

6 is a view confirming the production of CCR5/CXCR4 simultaneous knockout-induced pluripotent stem cells.

7 is a view confirming the pluripotency of CCR5/CXCR4 simultaneous knockout-induced pluripotent stem cells of the present invention.

8 is a diagram showing that CCR5/CXCR4 simultaneous knockout-induced pluripotent stem cells of the present invention can be differentiated into hematopoietic stem cells in vitro.

9 is a diagram showing that CCR5/CXCR4 simultaneous knockout-induced pluripotent stem cells of the present invention can be differentiated into hematopoietic stem cells in vivo.

10 is a diagram showing that the CCR5/CXCR4 simultaneous knockout-induced pluripotent stem cells of the present invention can be differentiated into hematopoietic stem cells in a serum-free/xeno-free/gene-free manner in vitro.

11 shows bone marrow, spleen, and peripheral blood when CCR5/CXCR4 simultaneous knockout-induced pluripotent stem cell-derived hematopoietic stem cells prepared in a serum-free / xeno-free / gene-free method are transplanted into a nude mouse. (Peripheral blood) is a diagram showing that it can be engrafted.

FIG. 12 shows that when CCR5/CXCR4 simultaneous knockout-induced pluripotent stem cells are differentiated into hematopoietic stem cells in vitro in a serum-free / xeno-free / gene-free manner, when treated with ginsenoside Rg1, a strong reactive oxygen species, the It is a diagram showing that engraftment ability can be dramatically improved.

The present inventors identified guide RNA target positions for CCR5 and CXCR4 genes, which can be targets of gene scissors, to treat HIV (Human Immunodeficiency Virus) infection using the CRISPR/Cas9 system, a programmable nuclease. . Accordingly, hematopoietic stem cells in which both CCR5/CXCR4 are knocked out can be prepared by cutting the guide RNA target site with gene scissors, and HIV infection can be effectively treated using this.

If it is described in detail as follows, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object, (a) reducing the expression level of CCR5 and CXCR4 in the isolated cell compared to the intrinsic expression level using a gene editing technique; and (b) culturing the cells isolated in step (a) using serum-free, xeno-free and gene-free methods to differentiate them into hematopoietic stem cells.

As used herein, the term "hematopoietic stem cell (HSC) is a cell that produces blood cells such as white blood cells, red blood cells, and platelets through self-replication and differentiation in the bone marrow. Hematopoietic stem cells, also called hematopoietic stem cells, are found in the bone marrow in adults. It is present in a small number of about 1% and is also present in a small number in peripheral blood.Because hematopoietic stem cells have the ability to self-replicate, even if blood cells are generated from hematopoietic stem cells and go out to the peripheral blood, the bone marrow cell fidelity (cell number change, morphology) of a normal person is ) is kept constant.In addition, hematopoietic stem cells have an instinct to visit the bone marrow, that is, to return home, and these characteristics enable transplantation of hematopoietic stem cells.On the other hand, the hematopoietic stem cells are cells with differentiation capacity, a type of stem cell It is also classified as

As used herein, the term "CCR5 (C-C chemokine receptor type 5)" is one of the transmembrane proteins present on the helper T cell surface, also called CD195. The CCR5 serves as a co-receptor when HIV is infected with helper T cells to help HIV infection, and HIV using CCR5 as a co-receptor is referred to as R5 type.

As used herein, the term "CXCR4 (C-X-C chemokine receptor type 4)" is one of the transmembrane proteins present on the surface of helper T cells, such as CCR5, also called CD184. The CXCR4 serves as a co-receptor when HIV is infected with helper T cells and helps in HIV infection, and HIV using CXCR4 as a co-receptor is referred to as an X4 type.

As used herein, the term "isolated cell" is not particularly limited, and specifically, it may be a cell whose lineage has already been specified, such as a somatic cell, a germ cell or a progenitor cell, and induced pluripotency. Stem cells, adult stem cells, bone marrow cells, mesenchymal stem cells, etc. may be stem cells with limited differentiation ability, specifically, the isolated cell of the present invention may be an induced pluripotent stem cell (iPSC) or a somatic cell, but is not limited thereto. . The isolated cells of the present invention may include both in vivo and ex vivo cells, and specifically, may be cells isolated in vivo.

In the present invention, "somatic cell" means all cells that have completed differentiation constituting animals and plants except for germ cells, and the "progenitor cell" refers to a cell corresponding to a progeny that expresses a specific differentiation trait when it is found to express a specific differentiation trait. Refers to parental cells that do not express , but have the differentiation fate (Fate).

In the present invention, "adult stem cells" refers to stem cells that appear at the stage of formation of each organ of the embryo or at the stage of adulthood through the development process, which is generally limited to cells constituting a specific tissue. Specifically, adult stem cells may be derived from the group consisting of breast, bone marrow, brain, spinal cord, umbilical cord blood, blood, liver, skin, gastrointestinal tract, placenta, and uterus.

In addition, in the present invention, "induced pluripotent stem cell (iPSC)" is an artificially created pluripotent stem cell by artificially expressing a specific gene to induce adult cells, which are mast cells, and is It is similar to pluripotent stem cells in many respects.

In the present invention, after knocking out the CCR5 and CXCR4 genes of induced pluripotent stem cells, they were differentiated into hematopoietic stem cells, and CCR5/CXCR4 knockout hematopoietic stem cells were prepared.

As used herein, the term "expression level is reduced compared to the endogenous expression level" means that the gene encoding the polypeptide is not expressed, or shows a specific decrease in gene expression compared to the natural state or state before modification, or is expressed even if functional means not to produce the corresponding polypeptide.

In addition, it includes not only that the gene encoding the polypeptide is completely inactivated, but also that the expression level is weakened or significantly lower than the intrinsic expression level and is not substantially expressed. Thus, gene inactivation is either complete (knockout) or partial (e.g., the product of a mutant gene that exhibits a partial decrease in the activity it affects or is a hypomorph that exhibits less than the intrinsic expression level of the gene. ) can be

Specifically, in the present invention, the inactivation of CCR5 or CXCR4,

1) deletion of a part or all of the polynucleotide encoding the protein;

2) modification of the expression control sequence to reduce the expression of the polynucleotide,

3) modification of the polynucleotide sequence on the chromosome such that the activity of the protein is weakened, or

4) It may be carried out using a combination thereof, etc., but is not particularly limited thereto.

1) In the method of deleting part or all of the polynucleotide encoding the protein, a polynucleotide encoding an endogenous target protein in a chromosome is replaced with a polynucleotide or a marker gene in which a part of the nucleic acid sequence is deleted through a vector for inserting the chromosome into a cell. It can be done by The "part" is different depending on the type of polynucleotide, but is specifically 1 to 300, more specifically 1 to 100, and more specifically 1 to 50.

Next, 2) the method of modifying the expression control sequence to reduce the expression of the polynucleotide is not particularly limited thereto, but the nucleic acid sequence is deleted, inserted, non-conservative or conservative to further weaken the activity of the expression control sequence. It can be carried out by inducing a mutation in the expression control sequence by substitution or a combination thereof, or by replacing the nucleic acid sequence with a weaker activity. The expression control sequence may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

In addition, 3) the method of modifying the polynucleotide sequence on the chromosome is performed by inducing a mutation in the sequence by deleting, inserting, non-conservative or conservative substitution of the polynucleotide sequence, or a combination thereof to further weaken the activity of the protein, or It can be carried out by replacing the modified polynucleotide sequence with a weaker activity, but is not limited thereto.

In the present invention, gene scissors can be used to reduce CCR5 and CXCR4 expression levels compared to intrinsic expression levels.

In the present invention, the term "gene scissors" is an artificial enzyme used to cut a specific DNA region by binding to an animal or plant gene, and may refer to a gene editing technology that solves a problem by removing a specific gene region. The gene editing technology includes a first-generation zinc finger nuclease (ZFN), a second-generation Transcriptor Activator-Like Effector Nucleases: TALEN, and a third-generation CRISPR (clustered regularly interspaced short palindromic repeats: CRISPR). ) may be included.

The zinc finger nuclease is a first-generation gene scissors in which a zinc finger and 3 to 4 nucleases are combined, and the nuclease uses a restriction enzyme called 'Fok1'.

Talen is a second-generation gene scissors derived from the plant pathogen Xanthmonas. Since the amino acid sequence constituting the Talen matches the base sequence of the DNA to be cut, if the amino acid sequence of the Talen is changed, the base sequence of the DNA to be bound can also be changed, which makes it much easier to customize the protein. 'Fokl' is used as an enzyme that cuts DNA like the first.

CRISPR is a type of immune system that is built with third-generation gene scissors to prevent viruses, the natural enemy of bacteria, and consists of a small palindromic structure. The most widely used CRISPR system is CRISPR/Cas9, where guide RNA complementarily binds to 20 base sequences of the target DNA, and Cas9 (CRISPR associated protein 9) recognizes a specific base sequence of DNA immediately following. cutting method. Recently, CRISPR/Cpf1 has been used as the fourth-generation gene scissors.

In addition, using a method in which the gene scissors and a template DNA similar to the target sequence are simultaneously introduced into cells, and the template DNA introduced into the target sequence site is knocked in by homologous recombination, the target sequence is knocked out can do it

Specifically, in the present invention, the expression level of CCR5 and CXCR4 using the CRISPR/Cas9 system including an expression vector containing both CCR5 and CXCR4 gene guide RNAs or a combination of expression vectors containing separately CCR5 and CXCR4 gene guide RNAs. may be reduced compared to the endogenous expression level, and the reduction may be to simultaneously knockout the CCR5 and CXCR4 genes, but is not limited thereto.

In addition, in the present invention, the step of reducing the expression level of CCR5 and CXCR4 in the cells isolated in step (a) compared to the intrinsic expression level may be characterized by using a composition for knockout of CCR5 and CXCR4, and the CCR5 and The composition for CXCR4 knockout includes (a) an expression vector comprising an isolated polynucleotide encoding a guide RNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto; and (b) an expression vector comprising an isolated polynucleotide encoding a guide RNA comprising the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence complementary thereto.

The guide RNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto targets the CCR5 gene, which is one of the co-receptors that help HIV infection, and the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence complementary thereto The guide RNA consisting of CXCR4 gene, which is another co-receptor, is targeted.

In the present invention, hematopoietic stem cells in which both the CCR5 and CXCR4 genes, which are co-receptors that help HIV infection, are knocked out were prepared. When transplanted into an HIV-infected patient, the hematopoietic stem cells are both CCR5 and CXCR4 genes knocked out. Since immune cells such as T cells can be made, it becomes resistant to HIV infection, and thus it is possible to treat AIDS, etc. caused by HIV.

As used herein, the term “RNA-guided nuclease” refers to a nuclease capable of recognizing and cleaving a specific position on a desired genome, particularly a nuclease having target specificity by a guide RNA. The RNA-guided nuclease is not limited thereto, but specifically Cas9 (CRISPR-Associated Protein 9) nuclease derived from CRISPR, which is a microbial immune system, and its variants, Cas9 nickase and Cpf1 nucleases.

As used herein, the term "Cas protein" is a major protein component of the CRISPR/Cas system, and is a protein that can act as an activated endonuclease. The Cas protein may form a complex with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to exhibit its activity.

The Cas9 nuclease causes a double strand break (DSB) by recognizing a specific nucleotide sequence in the genome of animal and plant cells including human cells. The double helix cutting includes cutting the double helix of DNA to make a blunt end or a cohesive end. DSBs are efficiently repaired in cells by homologous recombination or non-homologous end-joining (NHEJ) mechanisms. In this process, researchers can introduce desired mutations into target sites. The RNA-guided nuclease may be artificial or engineered non-naturally occurring.

The Cas9 nickase comprises at least one mutation in one of the catalytic domains of a Cas9 nuclease, wherein the at least one mutation is selected from the group consisting of D10A, E762A and D986A in the RuvC domain, or The one or more mutations are selected from the group consisting of H840A, N854A and N863A in the HNH domain.

In the present invention, the Cas9 nickase may have a D10A mutation, but is not limited thereto. The Cas9 nickase causes a single strand break unlike Cas9 nucleases. Thus, the Cas9 kinase requires two guide RNAs to function and function as a pair. The two guide RNAs direct sequence-specific binding of the CRISPR complex to each target sequence and direct cleavage of one strand of the DNA duplex near each target sequence, resulting in two nicks in different DNA strands. can induce

Cas protein or gene information can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI). Specifically, the Cas protein may be a Cas9 protein. In addition, the Cas protein is Staphylococcus genus, Streptococcus genus, Neisseria genus, Pasteurella genus, Francisella genus, Campylobacter) It may be a Cas protein derived from the genus, but the present invention is not limited to the examples described above.

In addition, the term "Cpf1 nuclease" of the present invention is derived from Francisella novicida , and may form a complex with crRNA to exhibit its activity. In addition, the structure of the Cpf1 protein is different from that of the Cas9 protein, and the length of the binding RNA is short, making it easy to manufacture and excellent in accuracy. The Cpf1 protein or gene information can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI).

The present inventors identified the target sequence of exon 3 of each of the CCR5 gene and the CXCR4 gene in order to prepare CCR5/CXCR4 simultaneous knockout hematopoietic stem cells for treating the human immunodeficiency virus (HIV) infection ( FIG. 1 ). The target position identified in the present invention has the nucleotide sequence of SEQ ID NO: 1 in the case of the CCR5 gene and the nucleotide sequence of SEQ ID NO: 2 in the case of the CXCR4 gene. Accordingly, the polynucleotide consisting of SEQ ID NO: 1 or a nucleotide sequence complementary thereto encodes a guide RNA for the CCR5 gene, and the polynucleotide consisting of SEQ ID NO: 2 or a nucleotide sequence complementary thereto encodes a guide RNA for the CXCR4 gene and can be used to prepare a guide RNA using the polynucleotide.

The expression vector of the present invention may additionally encode a Cas9 nuclease, a Cas9 nickase or a Cpf1 nuclease. That is, when the Cas9 protein or Cpf1 protein and the guide RNA exist together, the target DNA can be cut, so that the polynucleotide encoding the Cas9 protein or Cpf1 protein is included in the guide RNA and one vector. Alternatively, separate expression vectors encoding Cas9 protein or Cpf1 protein may be used. The vector may be a viral vector, a plasmid vector, or an Agrobacterium vector, but is not limited thereto. An expression vector comprising a polynucleotide encoding the guide RNA and/or a polynucleotide encoding a Cas9 protein or a Cpf1 protein may be prepared by performing a cloning method known in the art, and the method is particularly limited it's not going to be

The step of differentiating the isolated cells with reduced expression levels of CCR5 and CXCR4 compared to the intrinsic expression level of the step (a) of the present invention into hematopoietic stem cells differs depending on whether the isolated cells are induced pluripotent stem cells or somatic cells. method can be used.

First, when the isolated cells are induced pluripotent stem cells, hematopoietic stem cells can be differentiated in vitro or in vivo.

In the case of the in vitro method, hematopoietic stem cells can be prepared by culturing the isolated cells in a medium for hematopoietic stem cell differentiation. When the cells were cultured, it was confirmed that the hematopoietic stem cell differentiation efficiency was excellent (Example 3-1). In addition, another in vitro method is a method of co-culturing the isolated cells using OP9 cells, which are bone marrow stromal cells, as a feeder cell, and it was confirmed that hematopoietic stem cells can be prepared through the method. (Example 3-1).

In the case of the in vivo method, (i) forming a teratoma tissue by implanting the cells isolated in step (a) subcutaneously in a mouse; and (ii) isolating hematopoietic stem cells from the teratoma tissue formed in step (i).

Looking at an embodiment of the present invention, CCR5/CXCR4 simultaneous knockout-induced pluripotent stem cells obtained by accutase treatment to form teratoma tissue were mixed with Matrigel and subcutaneously injected into nude mice to form teratoma tissue. , Among them, CD34+ cells were isolated to prepare hematopoietic stem cells (Example 3-2).

In addition, when the isolated cells in which the expression levels of CCR5 and CXCR4 in step (a) are reduced compared to the intrinsic expression level are somatic cells, CCR5/CXCR4 knockout-induced pluripotent stem cells are prepared through reprogramming, and then the above-mentioned It is possible to differentiate the hematopoietic stem cells in an in vitro method or in an in vivo method, or may further include the step of inducing the somatic cells through direct reprogramming.

As used herein, the term "reprogramming" refers to a method of converting a target cell by controlling the global gene expression pattern of a specific cell. In other words, in the present invention, reprogramming refers to a method of artificially manipulating the fate of a cell to convert it into a cell having completely different characteristics, and for the purpose of the present invention, the reprogramming is a foreign gene or vector containing RNA or DNA. It may be carried out by introducing into the cell. For example, the reprogramming may include, but is not limited to, dedifferentiation of cells, direct reprogramming or direct conversion, or direct trans-differentiation.

As used herein, the term "reprogramming factor" refers to a gene (or a polynucleotide encoding the same) or protein that can be finally or partially introduced into differentiated cells to induce reprogramming. The reprogramming factor may vary depending on the target cell to induce reprogramming and the type of isolated cell from which reprogramming is induced. For example, in the case of producing hematopoietic stem cells, the reprogramming factor may include one or more factors selected from the group consisting of Lin28, Asc11, Pitx3, Nurr1, Lmx1a, Nanog, Oct3, Oct4, Sox2, Klf4 and Myc. , and in addition, it may include all factors known in the art to be capable of producing hematopoietic stem cells. In addition, direct reprogramming into hematopoietic stem cells can be induced by using the reprogramming factor. In the direct reprogramming methodology, there is a method of using a reprogramming genetic factor. A person skilled in the art can select an appropriate factor according to the target cell and the type of cell before reprogramming, which are all within the range known in the art. It is included in the scope of, it is not particularly limited to the type. Since reprogramming using reprogramming genetic factors induces conversion to target cells by regulating the entire gene expression pattern of cells, the reprogramming genetic factors are introduced into cells and culturing the cells for a certain period of time to obtain the desired type. It is possible to reprogram the initial cell to a target cell having the gene expression pattern of the cell.

As used herein, the term "direct reprogramming" is differentiated from the technology of producing induced pluripotent stem cells with pluripotency through the reprogramming process, and direct conversion to the desired target cell through reprogramming culture is used. it's technology Existing somatic cell nuclear transfer has the disadvantage of using an egg, so its applicability is low compared to other cell reprogramming technologies. In the case of using the induced pluripotent stem cell reprogramming technology, it naturally passes through pluripotent stem cells. There is a disadvantage in that the residual and safety must be verified. However, the present invention is expected to be able to provide an alternative that can overcome the problems of the above technology, such as production time, cost, efficiency, safety, etc., by directly producing hematopoietic stem cells, which are target cells, from initial cells through direct reprogramming. . For the purposes of the present invention, direct reprogramming may be used interchangeably with direct dedifferentiation, direct differentiation, direct conversion, direct cross-differentiation, cross-differentiation, and the like. In the present invention, direct reprogramming may refer to direct dedifferentiation or cross-differentiation into hematopoietic stem cells.

In the present invention, the cells isolated in step (a) can be differentiated into hematopoietic stem cells by culturing them by any one method selected from serum-free, xeno-free, and gene-free methods.

As used herein, the “free” method refers to a method of culturing cells without using a medium or a specific technique that does not contain a specific component or substance when culturing the cells.

The “serum-free” method may refer to culturing cells in a medium that does not contain serum and/or plasma. In one embodiment, it may include culturing the cells using a substance having a specified component (eg, albumin) that can replace it instead of serum (serum) of which the component is not specified.

The “xeno-free” method may mean culturing cells in a medium that does not contain animal-derived materials. In one embodiment, it may include culturing using a medium such as STEMdiff hematopoietic basal medium (STEMCELL) that does not contain animal-derived components instead of OP9 cells, which are mouse cells.

The “gene-free” method may refer to culturing cells without using a gene introduction method that may cause genetic changes in cells. In one embodiment, it may include culturing using a medium such as STEMdiff hematopoietic basal medium (STEMCELL).

In the present invention, when hematopoietic stem cells are produced using serum-free, xeno-free and gene-free methods, the consistency of experimental results is improved, unnecessary immune responses caused by animal-derived substances are excluded during transplantation in the body, and It can block the possibility of cancerous cells caused by

In the present invention, the cells isolated in step (a) can be differentiated by culturing in a medium containing an antioxidant.

The antioxidant may include without limitation a composition capable of enhancing the engraftment ability of differentiated cells. Specifically, selenium, vitamin E, catechin, lycopene, beta-carotene, coenzyme Q-10, EPA (eicosapentaenoic acid), DHA (docosahexanoic acid), tocopherol, vitamin C, L-lipoic acid, retinoic acid, vinpocetine, picamilon, It may be any one or more of quinic acid, adenine dinucleotide, acetyl-L-carnitine, dimethylanimo ethanol, C-MED 100, Trolox, GSH, protopanaxadiol, and ginsenoside. More specifically, it may be a ginsenoside.

The ginsenoside is included without limitation as long as it has antioxidant activity, for example, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, etc., but this is limited to this as an example doesn't happen Specifically, the ginsenoside may be ginsenoside Rg1, but is not limited thereto.

The antioxidant may be included in a concentration of more than 0 and 100 uM or less in the medium. Specifically, it may be 0.1 nM or more, 1 nM or more, 10 nM or more, 100 nM or more. Alternatively, 1 nM or more and 100 uM or less, 100 nM or more and 100 uM or less, 500 nM or more and 50 uM or less, but is not limited thereto.

The culture method may be used to enhance cell engraftment ability.

In the present invention, "engraftment ability" refers to the ability of the transplanted stem cells to replace the differentiated subsequent cells, the cells produced in the body after transplantation, or the injected cells to replace the lost or damaged cells. According to the enhancement of engraftment ability, for example, it can have advantages of reducing the number of effective transplanted stem cells, shortening the in vitro culture period of stem cells, increasing the safety of stem cells, and reducing manufacturing costs.

In one embodiment of the present invention, it was confirmed that the engraftment ability of the differentiated cells was improved by culturing the prepared pluripotent stem cells into hematopoietic stem cells in a serum-free, xeno-free and gene-free manner. Another aspect provides a method for improving the engraftment ability of differentiated cells by culturing stem cells in a serum-free, xeno-free, and gene-free manner.

The stem cells include all of pluripotent stem cells, pluripotent stem cells, and multipotent stem cells, and include induced pluripotent stem cells, adult stem cells, bone marrow cells, and mesoderm stem cells. As well as stem cells with limited differentiation ability, etc., as long as they have differentiation ability, they may be included without limitation.

Another aspect of the present invention comprises the steps of (a) differentiating into hematopoietic stem cells through direct reprogramming from the isolated somatic cells; and (b) reducing the CCR5 and CXCR4 expression levels of the hematopoietic stem cells of step (a) compared to the intrinsic expression level, providing a method for producing hematopoietic stem cells.

Isolated cells, hematopoietic stem cells, direct reprogramming, CCR5, CXCR4 and the step of decreasing compared to the intrinsic expression level is as described above.

Another aspect of the present invention provides a hematopoietic stem cell produced by the method for producing a hematopoietic stem cell.

The hematopoietic stem cells are as described above.

Another aspect of the present invention provides a pharmaceutical composition for treating or preventing HIV virus infection, comprising the hematopoietic stem cells as an active ingredient.

The HIV virus infection may specifically be acquired immune deficiency syndrome (AIDS), but is not limited thereto.

As used herein, the term “prevention” refers to any action that suppresses or delays the development of HIV infection by administering the composition.

As used herein, the term “treatment” refers to any action in which symptoms caused by HIV infection are improved or beneficially changed by administration of the composition.

The composition may include a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not inhibit the biological activity and properties of the injected compound without irritating the organism. The type of carrier usable in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in mixture of two or more.

The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient to the compound, for example, starch, calcium carbonate, sucrose, lactose. , gelatin, etc. may be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, etc. may be used.

The composition may be administered in a pharmaceutically effective amount.

The "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the individual type and severity, age, sex, type of virus infected, and drug. Activity, sensitivity to drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. For example, the composition or a pharmaceutically acceptable salt thereof may be administered at 0.0001 to 1000 mg/kg per day, preferably 0.001 to 100 mg/kg, respectively.

The administration means introducing the composition of the present invention to the patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, may be administered intranasally, but is not limited thereto.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into two to three times. When the two active ingredients are each single agent, the number of administrations may be the same or different. In addition, the composition of the present invention may be used alone or in combination with other drug treatments for the prevention or treatment of cancer. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

The subject means any animal, including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs that have or can develop cancer. . If the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject, the type of subject is included without limitation.

Another aspect of the present invention provides a cell therapy agent comprising the hematopoietic stem cells as an active ingredient.

The hematopoietic stem cells are as described above.

As used herein, the term "cell therapeutic" refers to cells and tissues manufactured through isolation, culture, and special manipulation from an individual, and is a drug used for treatment, diagnosis, and prevention purposes (US FDA regulations), and the function of cells or tissues is reduced. It refers to a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological properties of cells in other ways to restore them.

Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering the pharmaceutical composition or cell therapy to an individual in need thereof.

The “subject”, “administration”, “cancer”, and “treatment” are the same as described above.

Another aspect of the present invention is to improve the engraftment ability of stem cells by culturing stem cells in serum-free, xeno-free and gene-free methods. provide a way

Another aspect of the present invention provides a method for improving the engraftment ability of stem cells, comprising culturing the stem cells in a medium containing an antioxidant.

The above methods can be used in combination.

The stem cells may be specifically iPSC-derived cells, and more specifically iPSC-derived hematopoietic stem cells. In one embodiment, it may be a hematopoietic stem cell, prepared through the method for producing hematopoietic stem cells of the present invention, but is not limited thereto.

The antioxidant and stem cells were cultured in a serum-free, xeno-free and gene-free manner as described above.

When the culture method is used, the colony-forming ability of the stem cells, the engraftment ability of the stem cells, etc. may be improved. The culturing method may impart desirable characteristics of other stem cells.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These Examples are only for illustrating the present invention, and the scope of the present invention is not limited by the Examples and Experimental Examples.

Example 1: CRISPR/Cas9 construction for CCR5 and CXCR4 gene knockout

Example 1-1: CCR5 and CXCR4 target location selection and guide RNA production

In order to knock out the CCR5 and CXCR4 genes using the CRISPR/Cas9 system, guide RNAs targeting the CCR5 and CXCR4 genes were constructed. Candidate guide RNA sequences are listed in Table 1.

Specifically, a guide RNA was designed by targeting a specific sequence of exon 3 of the CCR5 or CXCR4 gene (Fig. 1a), and among the candidate guide RNA sequences, CCR5-1 and CXCR4-1, which are guide RNAs with excellent knockout efficiency, were used, respectively. It was selected as a guide RNA for knockout of CCR5 and CXCR4 genes ( FIGS. 1b and c ).

Then, using the pCas-guide vector, vectors containing the guide RNA for knockout of CCR5 and CXCR4 genes, respectively, were prepared. Specifically, 0.8 ul of two restriction enzymes (BamH1 and BsmB1) were mixed with 1 ug of the pCas-guide plasmid vector, respectively, and treated at 37° C. for 3 hours for cleavage, and self-linking through dephosphorylation of the cleaved vector was inhibited. 1ul of Antarctic phosphatase was added and incubated for 30 minutes at 37°C. Thereafter, the cleaved vector was purified using a gel purification column, eluted in 10 mM Tris buffer, and the synthesized guide RNA sequence was ligated to the cleaved vector.

Example 1-2: CCR5 and CXCR4 gene knockout confirmation using CRISPR/Cas9 system

In order to confirm that the CRISPR/Cas9 vector for CCR5 and CXCR4 knockout prepared in Example 1-1 can actually inhibit the expression of CCR5 and CXCR5 genes, the following experiment was performed.

First, in order to examine the conditions for introducing the CRISPR/Cas9 vector, the transformation efficiency was confirmed by introducing the vector into cells in various ways. Specifically, as a result of transformation using Lipopectamine 3000, Convoy, and an electroporation method, it was confirmed that the best efficiency was achieved when transformation was performed by the electroporation method (FIG. 2a). In addition, in order to establish electroporation conditions with excellent CRISPR/Cas9 vector introduction efficiency, transformation was performed under various conditions using a CRISPR/Cas9 vector containing GFP to confirm the GFP expression level ( FIGS. 2b and c ).

As a result, when Pulse voltage = 1450, Pulse width = 30, and Pulse No. = 2, it was confirmed that the CRISPR/Cas9 vector introduction efficiency was the best. In the following experiments, the electroporation method was performed under the above conditions.

After that, based on the experimental results, CRISPR/Cas9 for CCR5 and CXCR4 knockout prepared in Example 1-1 was introduced into cells by electroporation, and then the CCR5 and CXCR4 genes were knocked out. To amplify the CCR5 or CXCR4 gene portion, a PCR reaction was performed using the primers in Table 2 (FIG. 2a), and the knockout of the CCR5 and CXCR4 genes was confirmed through sequence analysis of the PCR product obtained therefrom.

Specifically, gDNA was isolated from cells into which CCR5 and CXCR4 knockout vectors were introduced using the AccuPrep Genomic DNA Extraction Kit (Bioneer). PCR amplification was performed using the gDNA isolated using AccuPower PCR Premix (Bioneer), CCR5 and CXCR4 forward and reverse primers (Table 2), and Genetouch Thermal Cycler (Hanzhou bioer technology) as a template. After PCR was completed, it was confirmed whether the gene was amplified through gel electrophoresis (Mupid) (FIG. 3a), and the PCR product was purified using MEGAquick-spin plus fragment DNA purification kit (iNtRON). The purified PCR product was sequenced in Macrogen (Seoul, Korea) together with the forward primer used for PCR (FIGS. 3b and c).

As a result of sequence analysis of the PCR product in which the CCR5 gene portion of the control and CRISPR/Cas9 vector-introduced cells were amplified, it was confirmed that a portion of the CCR5 gene was deleted when the CRISPR/Cas9 vector was introduced ( FIG. 3b ), and the control and As a result of sequence analysis of the PCR product in which the CXCR4 gene portion of the CRISPR/Cas9 vector was introduced, it was confirmed that a portion of the CXCR4 gene was deleted when the CRISPR/Cas9 vector was introduced ( FIG. 3c ). In addition, when the CRISPR/Cas9 vector was introduced, it was confirmed that the CXCR4 gene was knocked out monoallelic and biallelic ( FIG. 4 ).

Therefore, based on the above results, it can be seen that the CRISPR/Cas9 vector prepared in Example 1-1 knocked out the CCR5 and CXCR4 genes.

Example 2: Construction of iPSCs in which CCR5 and CXCR4 genes are knocked out

The following experiment was performed to construct induced pluripotent stem cells (iPSCs) in which CCR5 and CXCR4 genes were knocked out.

Specifically, iPSCs were prepared by introducing Yamanaka factors (Oct3/4, Sox2, Klf4 and c-Myc) into adult skin fibroblasts at Multiplicity of infection (MOI) 5, 10, and 20, and culturing them. The prepared iPSC had a typical colony shape, and it was confirmed that Oct4 and Nanog, which are pluripotency markers, were overexpressed ( FIG. 5 ).

In addition, qRT-PCR was performed to confirm the transcriptional expression levels of Oct4 and Nanog. Specifically, mRNA was isolated from the cell pellet using PureLink RNA Mini Kit (Invitrogen) to perform the qRT-PCR, and AccuPower cDNA was synthesized from the isolated mRNA using RT Premix (Bioneer). The cDNA, PowerUp SYBR Green Master Mix (Appliedbiosystems), Oct4 and Nanog forward and reverse primers (Table 3) and distilled water were mixed to make 20 ul, and PCR reaction was performed using Quant Studio3 (Appliedbiosystems), Oct4 and Nanog The relative fold values of the genes were calculated.

In addition, after introducing the CRISPR/Cas9 vector prepared in Example 1-1 into the iPSC, in order to confirm that the CCR5 and CXCR4 genes were knocked out, as in Example 1-2, the CCR5 and CXCR4 gene regions were PCR After amplification, the sequence of the amplified PCR product was analyzed.

As a result, it was confirmed that the CCR5 and CXCR4 genes were knocked out biallelic ( FIG. 6 ).

To determine whether the biallelic knockout iPSCs of the CCR5 and CXCR4 genes (CCR5 ^-/- CXCR4 ^-/- iPSCs) maintain the characteristics of induced pluripotent stem cells, CCR5 ^-/- CXCR4 ^-/- iPSCs are pluripotency marker The expression levels of Oct4, Sox2, Nanog, and TRA-1-60 were confirmed by immunostaining.

Specifically, the immunostaining method was performed as follows. First, Alexa Fluor 488 conjugated anti-Oct4 (Millipore), Cy3 to ^{the WT-iPSC and CCR5 -/-} CXCR4 ^-/- iPSC after fixation, permeabilization and blocking are completed for the purpose of the experiment Conjugated anti-Sox2 (Millipore), Alexa Fluor 488 conjugated anti-Nanog (Millipore), and Cy3 conjugated anti-TRA-1-60 (Millipore) primary antibodies (each 1:100) were treated at 4°C for one day. The next day, after washing with PBS, the stained cells were observed using a Nikon ECLIPSE Ti-U microscope (Nikon). Excitation/emission wavelengths were 488/525 nm for Alex Fluor 488 and 594/617 nm for Cy3. As a result of the immunostaining, it was confirmed that Oct4, Sox2, Nanog, and TRA-1-60 were overexpressed at the protein level in WT-iPSCs (FIG. 7a).

In addition, the ^{CCR5 - / - CXCR4 - / -} iPSC that was confirmed to be differentiated into 3-germ layer from the in vivo (Fig. ^{7b), CCR5 - / - CXCR4} - / - in which differentiation of iPSC cells typical markers of hematopoietic stem cells As it was confirmed through RT-PCR that CD34 is expressed ( FIG. 7c ), it can be seen that the CCR5/CXCR4 knockout iPSC prepared in the present invention has pluripotency even in vivo.

Example 3: Construction of CCR5 and CXCR4 knockout hematopoietic stem cells

Example 3-1: Production of hematopoietic stem cells using an in vitro method

The following experiment was performed to differentiate the CCR5/CXCR4 knockout iPSC (CCR5 ^-/- CXCR4 ^{-/- iPSC) prepared in Example 2 into hematopoietic stem cells in vitro.}

First, as a culture medium for differentiation into hematopoietic stem cells, iPSC spontaneous differentiation medium [DMEM/F12+GlutaMAX-I (Gibco) 500 ml + Primocin (Invivogen) 1 ml + Knockout SR (Gibco) 125 ml] and HSC culture medium [Stempro] -34 SFM SFM (1X) (Gibco) 500 ml + Stempro-34 Nutrient Supplement (Gibco) 13 ml + Glutamax-I (Gibco) 5 ml + Primocin (Invivogen) 1 ml + SCF (R&D) 100 ng/ml + IL-3 (Gibco) 50 ng/ml + GM-CSF (Gibco) 25 ng/ml] was mixed at 1:1 (v/v), and CCR5 ^−/− CXCR4 ^−/− iPSCs prepared in Example 2 were cultured (embryoid body) was cultured in the mixed medium for 2 weeks.

^{As a result, it was confirmed that the differentiation into CD34 +} cells expressing the hematopoietic stem cell marker CD34 was increased by about 20 times compared to the control (spontaneous differentiation) when differentiated with the mixed medium ( FIG. 8a ). It was confirmed that the differentiation of was enhanced in the mixed medium conditions.

^{In addition, as a result of differentiating CCR5 -/-} CXCR4 ^-/- iPSC using OP9 cells, which are bone marrow stromal cells, as a feeder cell, while having the form of hematopoietic stem cells (FIG. 8b), representative of hematopoietic stem cells It was confirmed that the marker CD34 was strongly expressed (FIG. 8c).

Therefore, it can be seen that the CCR5/CXCR4 knockout iPSC prepared in the present invention can be differentiated into hematopoietic stem cells using an in vitro method.

Example 3-2: Production of hematopoietic stem cells using in vivo method

In order to differentiate the CCR5 / CXCR4 knockout iPSC (CCR5 ^-/- CXCR4 ^-/- iPSC) prepared in Example 2 into hematopoietic stem cells in vivo, the CCR5 ^-/- CXCR4 ^-/- iPSC was injected into nude mice to form a teratoma, the teratoma was separated using a mechanical/chemical method, and a CD34 antibody was bound to the separated teratoma, followed by MACS sorting, followed by CD34 ⁺ cell differentiated CCR5 ^-/- CXCR4 ^-/- hematopoietic stem cells were obtained (FIG. 9a).

Specifically, for teratoma formation, about 1x10 ⁵ to 5x10 ⁵ CCR5/CXCR4 simultaneous knockout iPSCs were released with accutase, mixed with 100ul of matrigel, and implanted subcutaneously in Balb/c nude mice. After -12 weeks, it showed a teratoma formation rate of 100% (Wild type iPSC-2/2, simultaneous knockout iPSC-4/4) (FIG. 9b).

This is a dramatically improved result compared to the existing general teratoma formation rate. In general, the number of iPSCs injected into mice for teratoma formation is 1x10 ⁶ to 5x10 ^{6 In} spite of injecting a small amount of cells at a level of about 10% compared to 1x10 6 to 5x10 6 It showed a high teratoma formation rate. The above result is considered to be due to the synergistic effect of 100% Matrigel mixed with iPSCs immediately after using accutase, a non-strong protease, when removing iPSC colonies from the culture dish, and immediately after removing the cells.

In addition, the teratoma-derived CCR5/CXCR4 simultaneous knockout iPSC showed a CD34 expression rate at a similar rate to that of hematopoietic stem cells isolated from bone marrow ( FIG. 9c ). It can be seen that parent cells can be prepared.

Example 3-3: Production of hematopoietic stem cells using in vitro method (serum-free / xeno-free / gene-free method)

In order to use the CCR5/CXCR4 knockout iPSC (CCR5-/- CXCR4-/- iPSC) prepared in Example 2 as a cell therapy agent, hematopoietic stem cells (hematopoietic stem cells) in a serum-free / xeno-free / gene-free method ) to differentiate into the following experiment was performed.

First, the undifferentiated cells in the iPSC culture medium [DMEM/F12+GlutaMAX-I (Gibco) 500 ml + Primocin (Invivogen) 1 ml + Knockout SR (Gibco) 125 ml + bFGF 4ng/ml] for 5-7 days in an adherent state) The iPSCs were mechanically detached and seeded in a 12 well culture plate. A 12-well culture plate was used after coating for 2 hours at room temperature with matrigel (BD) diluted 1/45 in PBS. The number of iPSC aggregates seeded in a 12 well culture plate was about 10 (2-3/cm ² ) [(Manufacturer's conditions are 40-80 per well (10-20/cm ² )) . The reason for precisely controlling the seeding density of the iPSC aggregate to 2-3 pieces/cm ² is to prevent a sudden change in pH during the hematopoietic stem cell differentiation process to produce healthy hematopoietic stem cells that maintain their engraftment ability. One day later, as a medium for differentiation into hematopoietic stem cells, CCR5/CXCR4 knockout iPSCs were induced for 3 days in an adherent state in a differentiation medium mixed with STEMdiff hematopoietic basal medium (STEMCELL) 45ml, Primocin 90ul, and STEMdiff hematopoietic supplement A (STEMCELL) 225ul. did. From the 3rd to the 12th day of differentiation, the differentiation was induced with CCR5/CXCR4 knockout iPSCs in an adherent state in a differentiation medium mixed with 75ml of STEMdiff hematopoietic basal medium (STEMCELL), 180ul of Primocin, and 375ul of STEMdiff hematopoietic supplement B (STEMCELL). Hematopoietic stem cell differentiation was performed under 21% oxygen (normoixa) or 1-5% oxygen (hypoxia) for 12 days.

As a result, pure hematopoietic stem cells expressing 90% or more of CD34, a hematopoietic stem cell marker, were prepared (Fig. 10a). These hematopoietic stem cells could be differentiated into CD14-expressing monocytes (Fig. 10b, 10c) and CD44-expressing macrophages (Fig. 10d). When maintained in the iPSC iPSC aggregate seeding during differentiation in hematopoietic stem cells with low density (2-3 gae / cm ^2), which should make the differentiation into monocytes well on stem cell since compared to high density (10-20 gae / cm ²⁾ became (Fig. 10e).

Therefore, it can be seen that the CCR5/CXCR4 knockout iPSC prepared in the present invention can be differentiated into hematopoietic stem cells in a serum-free / xeno-free / gene-free manner.

Example 4: Verification of in vivo engraftment ability of hematopoietic stem cells prepared by serum-free / xeno-free / gene-free method

In order to utilize the hematopoietic stem cells derived from CCR5/CXCR4 knockout iPSC (CCR5-/- CXCR4-/- iPSC) prepared in Example 3-3 as a cell therapy agent, in vivo (bone marrow, spleen, peripheral blood) of a nude mouse The following experiment for transplanting hematopoietic stem cells (hematopoietic stem cells) was performed.

First, conditioning was performed to remove hematopoietic stem cells from the body of the mouse by injecting an anticancer agent (Busulfan, 50ug/g) intraperitoneally twice at 24 hour intervals. The next day, hematopoietic stem cells derived from CCR5/CXCR4 knockout iPSCs (CCR5-/- CXCR4-/- iPSCs) were intravenously injected into ^{5 x 10 4 cells per mouse.} After 8 weeks, mouse bone marrow, spleen, and peripheral blood were extracted and the presence of intravenously injected cells was checked by PCR.

As a result, it was confirmed that human cells having the same sequence as human CCR5 genomic DNA existed in mouse bone marrow (FIG. 11a). CCR5 knockout was also confirmed in these cells ( FIG. 11B ). The presence of human cells was also confirmed in spleen and peripheral blood (FIG. 11c).

Accordingly, it can be seen that the CCR5/CXCR4 knockout iPSC-derived hematopoietic stem cells prepared in the serum-free / xeno-free / gene-free method prepared in the present invention can be engrafted in vivo.

Example 5: Verification of enhancement of in vivo engraftment ability of hematopoietic stem cells by antioxidant

Example 5-1: Antioxidant screening

In order to enhance the engraftment ability of the hematopoietic stem cells derived from the CCR5/CXCR4 knockout iPSC (CCR5-/- CXCR4-/- iPSC) prepared in Example 3-3, the engraftment ability of hematopoietic stem cells during the differentiation process from iPSC to hematopoietic stem cells was evaluated. A screening for potent antioxidants was performed as follows.

First, undifferentiated in iPSC culture medium [DMEM/F12+GlutaMAX-I (Gibco) 500 ml + Primocin (Invivogen) 1 ml + Knockout SR (Gibco) 125 ml + bFGF 4ng/ml] attached for 5-7 days) The iPSCs were mechanically detached and seeded in a 12 well culture plate. A 12-well culture plate was used after coating for 2 hours at room temperature with matrigel (BD) diluted 1/45 in PBS. The number of iPSC aggregates seeded in a 12 well culture plate was about 10 (2-3 pieces/cm ² ).

One day later, as a medium for differentiation into hematopoietic stem cells, various concentrations of ginsenoside Rg1 (0nM, 1nM, 10nM, 100 nM, 1 uM, 10 uM) was added to induce differentiation of CCR5/CXCR4 knockout iPSCs in an adherent state for 3 days. From the 3rd to the 12th day of differentiation, 75ml of STEMdiff hematopoietic basal medium (STEMCELL), 180ul of Primocin, and 375ul of STEMdiff hematopoietic supplement B (STEMCELL) were mixed with various concentrations of ginsenoside Rg1 (0nM, 1nM, 10nM, 100nM, 1uM, 10uM) Differentiation was induced for 9 days in an adherent state of CCR5/CXCR4 knockout iPSCs in the differentiation medium. Hematopoietic stem cell differentiation was performed under 21% oxygen (normoixa) or 1-5% oxygen (hypoxia) for 12 days.

As a result, hematopoietic stem cells capable of forming hematopoietic colonies of various lineages in vitro were prepared, and the largest number of colonies was obtained at the concentration of Rg1 1uM (Fig. 12a). In a comparative experiment using various antioxidants other than Rg1 (Vitamin C (Vc) 50ug/ml, selenium 50ng/ml, melatonin 10uM), Rg1 1uM showed the best colony forming ability (Fig. 12b).

Therefore, it can be seen that the addition of Rg1 during the iPSC-derived hematopoietic stem cell differentiation process leads to qualitative improvement of the differentiated hematopoietic stem cells.

Example 5-2: Confirmation of enhancement of hematopoietic stem cell engraftment ability by the addition of antioxidants

In order to utilize the hematopoietic stem cells derived from CCR5/CXCR4 knockout iPSC (CCR5-/- CXCR4-/- iPSC) prepared in Example 5-1 as a cell therapy agent, in vivo (bone marrow, spleen, peripheral blood) of a nude mouse The following experiment for transplanting hematopoietic stem cells (hematopoietic stem cells) was performed.

First, conditioning was performed to remove hematopoietic stem cells from the body of the mouse by injecting an anticancer agent (Busulfan, 50ug/g) intraperitoneally twice at 24 hour intervals. The next day, hematopoietic stem cells derived from CCR5/CXCR4 knockout iPSCs (CCR5-/- CXCR4-/- iPSCs) were intravenously injected into ^{5 x 10 4 cells per mouse.} After 8 weeks, the peripheral blood of the mouse was extracted and the presence of intravenously injected cells was checked by flow cytometry.

As a result, in mouse peripheral blood, cells expressing human CD45 protein, which are common to human hematopoietic cells, were 28.7% (CCR5-/- CXCR4-/- iPSC-derived hematopoietic stem cell transplant group), 36.7% (Wild type iPSC-derived hematopoietic cells) hair cell transplant group), 74.4% (primary bone marrow-derived hematopoietic stem cell transplant group) was confirmed to be very high (FIG. 12c).

Therefore, it can be seen that the CCR5/CXCR4 knockout iPSC-derived hematopoietic stem cells prepared in the present invention show a very high engraftment ability in vivo.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

Claims (18)

1. (a) reducing the expression level of CCR5 and CXCR4 in the isolated cell compared to the intrinsic expression level using a gene editing technique; and

   (b) culturing the isolated cells of step (a) in serum-free, xeno-free and gene-free methods to differentiate them into hematopoietic stem cells; Including, hematopoietic stem cell production method.
2. The method of claim 1, wherein the cells isolated in step (a) are induced pluripotent stem cells (iPSCs) or somatic cells.
3. The method of claim 1, wherein the medium for culturing the cells in step (b) contains an antioxidant.
4. The method of claim 3, wherein the antioxidant is ginsenoside Rg1.
5. The method according to claim 4, wherein the ginsenoside Rg1 is contained in the medium at a concentration of 1 nM or more and 10 uM or less.
6. The method of claim 1, wherein the gene editing technique is any one of CRISPR, TALEN, or Zinc finger nucleases.
7. According to claim 1, wherein the step of reducing the expression level of CCR5 and CXCR4 in the cells isolated in step (a) compared to the intrinsic expression level is characterized in that using a composition for knockout CCR5 and CXCR4, hematopoietic stem cells manufacturing method.
8. The composition of claim 7, wherein the composition for knockout of CCR5 and CXCR4 is

   (a) an expression vector comprising an isolated polynucleotide encoding a guide RNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto; and

   (b) a method for producing hematopoietic stem cells comprising an expression vector comprising an isolated polynucleotide encoding a guide RNA consisting of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence complementary thereto.
9. The method of claim 8, wherein the expression vector comprises a polynucleotide encoding any one of Cas9 nuclease, Cas9 nickase, or Cpf1 nuclease. .
10. (a) differentiating the isolated somatic cells into hematopoietic stem cells through direct reprogramming; and

    (b) reducing the CCR5 and CXCR4 expression levels of the hematopoietic stem cells of step (a) compared to the intrinsic expression level;

    Including, hematopoietic stem cell production method.
11. A hematopoietic stem cell prepared by the method of any one of claims 1 to 10.
12. A pharmaceutical composition for the treatment or prevention of HIV virus infection, comprising the hematopoietic stem cells of claim 11 as an active ingredient.
13. A cell therapy product comprising the hematopoietic stem cells of claim 11 as an active ingredient.
14. A method for improving the engraftment ability of stem cells by culturing stem cells in serum-free, xeno-free and gene-free methods.
15. 15. The method of claim 14, wherein the method improves the consistency of experimental results, excludes unnecessary immune responses caused by animal-derived substances during transplantation in the body, and blocks the possibility of cancerous cells caused by foreign genes.
16. A method for improving the engraftment capacity of stem cells, comprising culturing the stem cells in a medium containing an antioxidant.
17. The method of claim 16, wherein the antioxidant is ginsenoside Rg1.
18. The method of claim 16, wherein the stem cells are iPSC-derived hematopoietic stem cells.

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