WO2018151514A1 - Composition for preventing or treating bone diseases which has excellent bone regeneration effect - Google Patents

Abstract

The present invention relates to a composition for preventing or treating bone diseases, comprising a BMP-2-encoding gene and HSV-tk-encoding gene, and as an active ingredient a stem cell into which a dual kill switch expression vector in which an HGPRT-encoded gene is knocked out is introduced or a cell differentiated from the stem cell, wherein the bone regeneration effect is realized by a BMP-2 growth factor and at the same time, apoptosis may also be dually controlled by the dual kill switch.

Description

A composition for preventing or treating bone diseases with excellent bone regeneration efficacy

The present invention relates to a composition for preventing or treating bone diseases excellent in bone regeneration efficacy, and implements a bone regeneration effect through a BMP-2 growth factor and at the same time provides a double safety device for controlling cell death through a double kill switch. Include as an active ingredient.

Bones, which support the soft tissues and weight of the body and protect internal organs from external shocks, are not only structurally supporting muscles or organs, but also an important part of the body that stores essential minerals such as calcium, phosphorus and magnesium in the body.

The bones of the grown adult are balanced by the process of repeating the generation and absorption process of removing old bones and replacing them with new bones. This is called bone remodeling (Yamaguchi A. et al., Tanpakushitsu Kakusan). Koso., 50 (6 Suppl); 664-669, 2005). This bone circulation is essential to repair and maintain the microscopic damage of bone caused by growth and stress. In adults, about 10% to 30% of the skeleton is reshaped every year through remodeling of bone resorption-osteoblasts.

Osteoblast formation involves osteoblasts that produce bone and osteoclasts that destroy bone, and bone homeostasis is maintained through close interaction between them. For example, osteoblasts are in the body by controlling the differentiation of osteoclasts responsible for bone resorption through the secretion of a substance, such as RANKL (receptor activator of nuclear factor- _К B ligand), and their induction of receptor OPG (osteoprotegerin) Maintain goal homeostasis.

In the past, research has focused mainly on bone minerals, namely calcium and phosphorus metabolic abnormalities, in the case of bone homeostasis not maintained due to physical influences, hormonal systems, or the treatment of bone diseases associated with bone tissue damage. No progress has been made in identifying the mechanism.

In general, a diet containing calcium is recommended for the treatment and prevention of osteoporosis, and estrogen or vitamin D administration is recommended for postmenopausal women. In addition, bisphosphonate-based drugs such as Fosamax (component name: alendronate) and Actonel (component name: risedronate) are attracting attention as new alternative therapeutics as bone resorption inhibitors that inhibit osteoclasts and induce death. .

However, calcium adjuvant widely used in the treatment of bone diseases suppresses secretion of parathyroid hormone and prevents bone loss due to bone resorption, but it is known that individual differences in maintenance of bone mass are severe (Heandy RP principles of bone biology, Academic press, 1007). -1017, 1996). In addition, hormone therapy with estrogen or calcitonin has been reported to increase bone density and reduce the incidence of rectal cancer, but side effects such as breast cancer, myocardial infarction and venous thrombosis have been reported (Nelson, HD et al., JAMA, 288: 872-881, 2002; Lemay, A., J. Obstet. Bynaecol. Can., 24: 711-7152-3).

In the case of bisphosphonate preparations, the incidence of jawbone necrosis, severe atrial fibrillation, incapacitation of bones or joints, or musculoskeletal pain in recent patients has been increasing yearly (Coleman RE., Br J Cancer, 98: 1736-1740 ( Therefore, there is a need for the development of a new bone disease treatment agent that can effectively suppress bone resorption while having fewer side effects.

After fracture, various growth factors cause damage and distant mesenchymal stem cells to become mature osteocytes through recruitment / differentiation and bone regeneration occurs through the generation of new blood vessels and bone tissue.

At this time, BMP-2, a protein that plays a key role in the recruitment and differentiation of stem cells, is known as a growth factor that plays the most crucial role in bone regeneration. As a growth factor, BMP-2 growth factor, a form of recombinant protein, is used in the form of an injection in clinical trials because of its bone regeneration effect, but more than 80% of the growth factor is released during the initial injection, and thus it is not effectively used.

Existing treatment methods for the treatment of bone regeneration include surgical treatment, treatment using biomaterials / tissue engineering, treatment using stem cells / growth factors. Autogenous bone grafts in bone grafts require donor injury and repetitive surgery. Allogeneic bone xenograft grafts may result in insufficient bone regeneration or complications such as infections.

Biomaterial scaffold has a disadvantage in that when bone defects are large due to lack of osteoinduction, the bone regeneration effect is weak and stem cells or growth factors are required. Currently, bone regeneration therapy using stem cells / growth factors is insignificant and requires the development of functional cell therapies that can improve bone regeneration through the combination of stem cells and growth factors.

On the other hand, cell therapy is also a problem that needs to be solved because of the possibility of differentiating into unwanted cells in vivo to inhibit the function of the tissue or develop into a malignant tumor.

The present invention is to solve the above problems of the prior art, an object of the present invention is to provide a composition for the prevention or treatment of bone diseases having an excellent biosafety while improving the efficacy of bone regeneration.

However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

According to an embodiment of the present invention, a stem cell comprising a BMP-2 encoding gene and an HSV-tk encoding gene, into which a dual kill switch expression vector to which the HGPRT encoding gene is knocked out is introduced, or Provided is a composition for preventing or treating bone diseases, comprising the cells differentiated from the stem cells as an active ingredient.

According to one side, the stem cells may be embryonic stem cells (ESC, Embryonic stem cells) or mesenchymal stem cells (MSC, Mesenchymal stem cells).

According to one side, the cells differentiated from the stem cells may be fibroblast (osteoblast) or osteoblast (osteoblast).

According to one side, the fibroblasts may be teratoma-derived fibroblast (TDF).

According to one side, the bone disease may be one or more selected from the group consisting of bone defects, osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, osteoplasia and osteomalacia.

According to one side, the composition ALP (Alkaline phosphatase), IBSP (Integrin binding sialoprotein), RUNX2 (Runt-related transcription factor 2), OSX (Osterix), SPP1 (Secreted phosphoprotein 1) and OCN (Osteocalcin) group One or more marker expressions selected from can be increased.

According to one side, the composition may further comprise a scaffold.

According to one side, the support may be made of polycaprolactone (PCL, Polycaprolactone) or biphasic calcium phosphate (BCP).

According to another embodiment of the invention, preparing a vector comprising a BMP-2 coding gene and HSV-tk coding gene; Knocking out the HGPRT encoding gene in the vector; And introducing the vector into stem cells or cells differentiated from the stem cells. There is provided a method of preparing a composition for preventing or treating bone diseases.

Since the composition for preventing or treating bone diseases of the present invention includes stem cells into which the BMP-2 gene has been introduced, the BMP-2 growth factor may be generated per se, and thus, an excellent bone regeneration effect may be realized.

In addition, the stem cells include the HSV-tk gene, and knock out the HGPRT encoding gene to implement a double kill switch, thereby preventing cancer cell transformation by autoproliferation, etc., and improving human safety.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 shows the morphology before and after Cre treatment of cells and BMP-2 and HSV-tk gene expression diagram according to an embodiment of the present invention.

2 is a graph showing the BMP-2 release behavior of the cell line according to an embodiment of the present invention.

Figure 3 is a graph showing the ALP activity of the cell line according to an embodiment of the present invention.

Figure 4 is a graph showing the ALP activity of the TDF cell line injected with BMP-2 from the outside.

5 is a graph showing BMP-2 release behavior during bone differentiation of cell lines according to an embodiment of the present invention.

Figure 6 is a graph showing the level of gene expression related to bone differentiation according to an embodiment of the present invention.

Figure 7 is a graph showing the calcium deposition of the cell line according to an embodiment of the present invention.

8 is a graph showing the mineral deposition of the cell line according to an embodiment of the present invention.

9 is an X-ray image showing the bone formation efficacy for the femoral bone defect animal model of the cell line according to an embodiment of the present invention.

Figure 10 is a micro-CT showing the bone formation efficacy of the cell line according to an embodiment of the present invention.

FIG. 11 is a graph comparing H & E tissue staining results and bone loss size of bone cell deficient animal models of cell lines according to an embodiment of the present invention.

Figure 12 is an image showing the cell morphology according to the concentration and time of gancyclovir treatment of the cell line according to an embodiment of the present invention.

13 is a graph showing the number of cells with gancyclovir treatment concentration and time.

Figure 14 is an image of the shape of the cell line according to an embodiment of the present invention.

15 is a graph showing the BMP-2 release behavior of the cell line according to an embodiment of the present invention.

16 is a graph showing the level of gene expression related to bone differentiation according to an embodiment of the present invention.

Figure 17 is a graph showing the calcium deposition of the cell line according to an embodiment of the present invention.

18 is a graph showing the mineral deposition of the cell line according to an embodiment of the present invention.

19 is an H & E tissue staining result showing bone formation efficacy for the skull bone defect animal model of the cell line according to an embodiment of the present invention.

20 is an image showing the results of resistance test for 6-TG drug of the cell line according to an embodiment of the present invention.

21 is an image showing the cell killing effect of the aminopterin drug against the cell line according to an embodiment of the present invention.

22 is an image showing the expression level of the bone regeneration marker of the cell line according to an embodiment of the present invention.

Figure 23 is a graph comparing the BMP-2 release behavior with or without lgK attachment.

24 is a graph comparing HGPRT expression levels of the control group and the double kill switch group after HGPRT gene editing.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in the drawings denote like elements.

Various modifications may be made to the embodiments described below. The examples described below are not intended to be limited to the embodiments and should be understood to include all modifications, equivalents, and substitutes for them.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

In addition, in the description with reference to the accompanying drawings, the same components regardless of reference numerals will be given the same reference numerals and redundant description thereof will be omitted. In the following description of the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

According to an embodiment of the present invention, a stem cell comprising a BMP-2 encoding gene and an HSV-tk encoding gene, into which a dual kill switch expression vector to which the HGPRT encoding gene is knocked out is introduced, or Provided is a composition for preventing or treating bone diseases, comprising the cells differentiated from the stem cells as an active ingredient.

The BMP-2 is a kind of bone morphogenetic protein that is involved in the healing of cartilage-resistant membrane fractures and promotes bone growth as well as essential for natural regeneration reactions. Compared to the case of injecting BMP-2 from the bone disease treatment efficacy can be improved.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a protein that inhibits apoptosis, and removing HGPRT encoding genes can be a double safeguard against apoptosis control failure due to loss of HSV-tk. That is, by inserting the HSV-tk coding gene into the expression vector, not only a single kill switch can be implemented, but in addition, the HGPRT coding gene can be knocked out to implement a dual kill switch. Specifically, when the double kill switch expression vector is introduced into a cell line, apoptosis may be induced by treating a drug such as aminopterin. Therefore, the composition of the present invention can effectively control the cell death can prevent side effects of stem cell therapeutics such as aberrant proliferation, malignant tumors.

The BMP-2 introduced cell line may be a stem cell, and specifically, the stem cell may be an embryonic stem cell (ESC) or a mesenchymal stem cell (MSC). The stem cells may perform their own osteogenic function separately from the bone formation caused by BMP-2, but when applied together with BMP-2, more effective bone formation effect may be realized.

In addition, the cells differentiated from the stem cells may be fibroblast (osteoblast) or osteoblast (osteoblast). In this case, the fibroblasts may be teratoma-derived fibroblasts (TDF).

That is, the cells into which the BMP-2 coding gene is introduced may be fibroblasts derived from teratomas formed from embryonic stem cells, and osteoblasts differentiated from embryonic stem cells, but are not limited thereto.

As used herein, the term “teratoma” is a kind of tumor composed of various cells and tissues such as skin cells, muscle cells, and nerve cells, whereas a general tumor is composed of a single cell. It can be formed by injecting. By separating fibroblasts generated from the teratoma and using them as cell lines, the expression efficiency of BMP-2 can be improved. In addition, the fibroblasts can be differentiated into osteoblasts can implement osteogenic function.

On the other hand, since the stem cells or cells differentiated from the stem cells have excellent differentiation in and of themselves, may not only function as a therapeutic agent but also pose a risk of development into cancer cells.

Accordingly, by introducing a suicide gene such as HSV-tk as well as the BMP-2 gene into the cell line, the cell line can be suppressed from developing into cancer cells.

At this time, an internal ribosome entry site (IRES) gene may be inserted between the BMP-2 gene and the HSV-tk gene. Using the IRES gene it is possible to express both the BMP-2 gene and HSV-tk gene with one promoter. Specifically, the BMP-2 gene, IRES gene and HSV-tk gene may be composed of the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

In addition, the BMP-2 gene may be a gene encoding the Lgk peptide at the 5 'end. The Lgk peptide is a leader peptide that induces extracellular release of BMP-2 protein generated in cells, and can improve bone formation efficacy by the BMP-2 protein. The BMP-2 gene into which the Lgk peptide coding gene is introduced may consist of a nucleotide sequence represented by SEQ ID NO: 4.

The HSV-tk gene phosphorylates drugs such as acyclovir and gancyclovir, and phosphorylation of these drugs can induce cell death by inhibiting DNA synthesis by DNA polymerase. Accordingly, cancer cellization of the cell line can be prevented by administering a drug such as acyclovir or gancyclovir at a time point at which bone formation by BMP-2 is sufficiently induced in the cell line. In other words, these HSV-tk genes can act as kill switches for cell lines.

Meanwhile, the BMP-2 coding gene and the HSV-tk coding gene may be inserted into one expression vector, and the genes may be expressed by transfection of the expression vector into a host cell. For example, the expression vector may be an adenovirus expression vector, an adeno-associated virus, an retrovirus vector or a plasmid, but is not limited thereto.

The bone disease to be prevented or treated of the composition may be a disease associated with bone mass to a decrease in bone density, specifically selected from the group consisting of bone defects, osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, osteoplasia and osteomalacia It may be one or more, but is not limited thereto.

Accordingly, the composition can increase the expression of genes or proteins involved in improving bone mass to bone density. Specifically, the composition is selected from the group consisting of Alkaline phosphatase (ALP), Integrin binding sialoprotein (IBSP), Run-related transcription factor 2 (RUNX2), Osterix (OSX), Secreted phosphoprotein 1 (SPP1), and Osteocalcin (OCN). It is possible to increase the expression of one or more markers, but is not limited thereto.

The composition may exhibit excellent bone formation ability by itself, but may further include a scaffold to further improve its efficacy. At this time, the support may be made of polycaprolactone (PCL, Polycaprolactone) or biphasic calcium phosphate (BCP), but is not limited thereto. The support may perform a function of fixing the cell line included in the composition to the implanted position.

On the other hand, according to another embodiment of the present invention, preparing a vector comprising a BMP-2 coding gene and HSV-tk coding gene; And introducing the vector into stem cells or cells differentiated from the stem cells. There is provided a method of preparing a composition for preventing or treating bone diseases.

The functions of the BMP-2 coding gene and the HSV-tk coding gene, the method of introducing them into the cell line, and the types of the bone diseases are as described above. On the other hand, the method of knocking out the HGPRT encoding gene may use a known gene removal method, for example, TAL-nuclease, meganuclease, zinc-finger nuclease (ZFN), or RNA-induced It may be performed using endonucleases. According to one embodiment of the present invention, the Cas9 / CRISPR method may be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1 Establishment of TDF Cell Lines

10 ⁶ embryonic stem cells (WA01 male embryonic stem cells, WiCell research institute) were mixed with 30% matrigel (BD biosciences), and then subcutaneously injected into SCID (severe combined immunodeficiency) mice. After 6 weeks, the formed teratoma was isolated and ground to 1 mm size, followed by 10% FBS, 1% non-essential amino acids, 1% penicillin-steptomycin, 2mM glutamax and Incubated in DMEM culture medium containing 55μΜ β-mercaptoethanol (β-mercaptoethanol). Fibroblasts (TDF, Teratoma-derived fibroblast) grown from teratoma tissues during culture were separated by trypsin and passaged to establish cell lines.

Example 2: Construction of BMP-2 Coding Gene and HSV-tk Coding Gene Insertion Vector

PL453 vector containing CAG promoter-loxP-neo-loxP was digested with NotI restriction enzyme and blunt end was formed using T4 DNA polymerase. Plasmid DNA containing the HSV-tk gene was digested with BglII / NcoI restriction enzyme, blunt ends were formed using T4 DNA polymerase, and then inserted into the vector.

Thereafter, the vector was cleaved with BamHI restriction enzyme to form a blunt end using T4 DNA polymerase, and then the BMP-IRES portion was amplified by PCR and inserted into the vector to insert the BMP-2 gene and the HSV-tk gene. The generated vector.

Example 3: Introduction of Vectors into Cell Lines

After dispensing the TDF cell line of Example 1 to 2 × 10 ⁶ cells in a ⁶⁰ phi dish, the vector of Example 2 was transfected. After 48 hours of transfection, cells without neoR gene were killed by treatment with neomycin for 5 days, and cells into which BMP-2 gene was introduced were selected.

Thereafter, the plasmid DNA containing pCAG-Cre was transfected into the cells so that the BMP-2 and HSV-tk genes were expressed by reacting the loxP gene with the Cre protein (FIG. 1). The expression of BMP-2 was confirmed by ELISA analysis, and the results are shown in FIG. 2. Referring to Figure 2, two treatments with Cre was confirmed that the release of extracellular BMP-2 of about 2ng / ㎖.

Example 4 Evaluation of Initial Bone Formation Induction Capacity

In order to evaluate the in vitro early bone formation ability of the cell line according to Example 3, the activity level of ALP (Alkaline phosphatase), an index of the early stage of bone formation was measured.

In order to measure the ALP activity of the TDF cell line of Example 3 and the normal TDF cell line not expressing BMP-2, a group of cells cultured in an Osteogenesis induction medium (OIM) and a general growth culture (GM, Growth medium) was divided into groups incubated for 3 days, 7 days and then each ALP activity was measured.

Specifically, the samples after 3 and 7 days of culture were washed twice with PBS buffer and then dissolved in a cell lysis buffer to which 0.1% Triton X-10 was added. The lysed cells were centrifuged at 4 ° C. and 13000 rpm for 30 minutes, and then the supernatant was recovered and quantitated by Bradford assay. The same amount of protein was used at 405 nm using ALP kit (AnaSpec) at 405 nm. Activity was measured and the results are shown in FIG. 3.

Referring to FIG. 3, 7 days after the induction of bone formation, the ALP activity of the cell line of Example 3 was observed to be about 2 times higher than that of the control group, and the cell line of Example 3 exhibited ALP activity even in normal growth culture. It was. Through this, it can be seen that the TDF cell line introduced with BMP-2 can implement excellent initial osteogenic induction ability.

On the other hand, in order to evaluate the osteogenic induction ability when injecting BMP-2 protein from the outside compared to the TDF cell line of Example 3, commercially available recombinant BMP-2 protein was injected into a normal TDF cell line and the same experiment as above It carried out, and the result is shown in FIG.

Referring to FIG. 4, 7 days after the induction of bone formation, all experimental groups treated with BMP-2 at 1 ng / ml, 2 ng / ml, and 5 ng / ml were statistically significant compared to the control group without BMP-2. ALP activity could not be observed. Considering that the concentration of BMP-2 released from the TDF cells of Example 3 is 2 ng / ml as described above, when the TDF cell line expressing the BMP-2 protein directly therein injects BMP-2 from the outside, It can be seen that it can implement a far superior bone formation induction ability.

Example 5 Evaluation of BMP-2 Release Behavior During Bone Formation

In order to confirm that the TDF cell line of Example 3 maintains BMP-2 release even during the bone formation process, 3 and 7 days after the incubation of the TDF cell line of Example 3 in OIM-induced culture medium (OIM) The BMP-2 release behavior was analyzed by ELISA analysis, and the results are shown in FIG. 5.

Referring to Figure 5, it can be seen that improved BMP-2 release in the TDF cell line of Example 3 compared to the TDF cell line without any treatment at the 3 and 7 days of culture. Therefore, it can be seen that the TDF cell line of Example 3 can continuously maintain BMP-2 release during bone formation.

Example 6: Determination of Bone Formation Related Marker Expression Levels

Expression of Alkaline phosphatase (ALP), Integrin binding sialoprotein (IBSP), and bone differentiation-related transcription factors, RUNX2 (Runt-related transcription factor 2) and OSX (Osterix), in the TDF cell line of Example 3 To confirm the increase, induce osteoblast differentiation of the TDF cell line and TDF cells without BMP-2 introduction, and after 3 days, extracting their entire RNA using Trizol reagent (Invitrogen) and expressing the genes of the markers. The level was measured.

Specifically, each cultured cell was washed twice with PBS, lysed with 1 ml of Trizol reagent, and then stirred by adding 200 µl chloroform and centrifuged at 4 ° C and 12000 rpm for 20 minutes to separate the supernatant. 500 μl of isopropanol was added to the separated supernatant, followed by stirring, followed by centrifugation at 4 ° C. and 12000 rpm. The pellet, except the supernatant, was washed three times with 70% ethanol and RNA was isolated.

5 μl of each RNA was prepared, added to the RT-PCR amplification kit, and reacted at 45 ° C. for 60 minutes to prepare cDNA. The prepared cDNA was amplified by real-time PCR using primers specific for ALP, OCN (Osteocalcin) and OPN (Osteopontin), respectively.

Specifically, 10 μl of 2X SYBR Green Reagent (Roche) and each primer (0.5 pmol / μl) were added to the same amount of cDNA, and then amplified by reacting 40 times at 95 ° C. for 30 seconds and 60 ° C. for 1 minute. PCR results are shown in FIG. 6.

Referring to Figure 6, it can be seen that the TDF cell line of Example 3 increases the gene expression levels of the bone formation markers ALP, IBSP, RUXN2 and OSX, which is effectively BMP-2 introduced TDF cell line Imply that it can be induced.

Example 7 Late Bone Formation Induction Evaluation

In order to evaluate the late bone formation ability of the TDF cell line of Example 3, the TDF cell line of Example 3 and a normal TDF cell line that does not express BMP-2 were respectively formed in bone formation induction medium (OIM) or normal growth culture ( GM) and calcium and mineral deposition were measured after incubation for 10 to 12 days.

Calcium deposition was measured with QuantiChrom ^™ Calcium Assay Kit (DICA-500). Specifically, samples were taken 10 days after induction of bone differentiation, washed twice with PBS buffer, treated with 0.6N HCl, and stored at 4 ° C. for 24 hours. Calcium deposited on the cells was measured at 612 nm using the calcium assay kit, and the results are shown in FIG. 7.

Meanwhile, mineral deposition was measured using Arizarin red S staining solution (Millipore). Specifically, the samples 10 days after the induction of bone differentiation were washed twice with PBS buffer, and the cells were fixed for 15 minutes with 4% paraformaldehyde. The fixed solution was removed, washed with distilled water, and then added to the Arizarin red S stain and stored at room temperature for 20 minutes. After the dyeing was completed and washed three times with distilled water to confirm the color change and at the same time the optical density (OD) at 570nm was measured, the results are shown in FIG.

Referring to FIGS. 7 and 8, the TDF cell line of Example 3 showed more than 4 times calcium deposition and more than 2 times mineral deposition compared to the control group, and it can be seen that it is excellent in inducing late bone formation.

Example 8: Assessment of Bone Formation in Animal Models

In order to confirm the bone regeneration effect of the TDF cell line of Example 3 in the femoral bone defect animal model, a bone defect model was performed through segmental resection of the iliac bone after surgically exposing the thigh of the rat at 7 weeks of age. Was produced. At this time, the size of the bone defect was produced to 7 mm so that coalescence does not occur after 4 weeks and 8 weeks of partial excision.

The TDF cells of Example 3 and the control TDF cells without BMP-2 were mixed with 5 × 10 ⁵ polycaprolactone (PCL, Polycaprolactone) scaffolds and incubated at 37 ° C. for 24 hours. The rat was introduced into a bone defect site. Thereafter, the surgical site was sutured and finished by fixing with an external fixation device, and bone formation was observed using X-rays at one week intervals, and the results are shown in FIG. 9.

Referring to FIG. 9, compared to the group injected with only PCL scaffold after 2 weeks of bone defect, both the group injected with control TDF cells and the group injected with TDF cells of Example 3 showed an increase in bone formation. have. In particular, at 4 weeks after the bone defect, the TDF cells of Example 3 showed more effective union of the bone defect region than the control TDF cells.

In addition, after 4 weeks of bone defect, the iliac bone defect site of the rat was photographed by micro-CT, and the bone formation site was measured and compared. Referring to FIG. 10, the group introducing the TDF cell line of Example 3 exhibited significantly improved bone formation ability compared to the group introducing only the PCL support and the control group introducing the TDF cell line.

Meanwhile, in order to confirm the bone regeneration effect of the TDF cell line of Example 3 in the skull bone defect animal model, a bone defect model was produced through segmental resection after surgically exposing the skull of an 8-week-old nude mouse. It was. At this time, the bone defect size was 4 mm to prevent fusion after 4 and 8 weeks of partial ablation.

TDF cells of Example 3 and control TDF cells without BMP-2 were mixed with 5 × 10 ⁵ or more calcium phosphate (BCP) scaffolds and incubated at 37 ° C. for 24 hours. It was then introduced into the bone defect site of the rat. After 3 weeks, the bone defects were collected and bone formation was evaluated using the H & E tissue staining test. The results are shown in FIG. 11, and the osteocalcin and Lamin A / C markers were fluorescently stained. The confirmed result is shown in FIG. . Referring to Figure 11, the TDF cell line introduction group of Example 3 shows an excellent bone formation effect compared to the control TDF cell line introduction group, it can be seen that the most reduced the defect site size. In the TDF cell line introduction group of Example 3, it was confirmed that the expression of osteocalcin and lamin A / C, which are bone regeneration markers, were more prominent than the control TDF cell line introduction group, which is a bone regeneration effect in the TDF cell line introduction group of Example 3 Suggests that it is better.

Example 9: Apoptosis Evaluation

In order to evaluate the cell death according to HSV-tk gene expression according to the TDF cell line according to Example 3, 0 μg / ml, 50 μg / ml and 500 μg of gancyclovir in the cell line of Example 3 The cells were treated at a concentration of / ml and observed and analyzed for 72 to 124 hours, respectively, and are shown in FIGS. 12 and 13, respectively.

Referring to FIGS. 12 and 13, it was confirmed that cell growth was inhibited depending on the concentration and time of Gancyclovir treatment, indicating that the kill switch of the cell was activated according to HSV-tk gene expression. Through this, it can be seen that the TDF cell line of Example 3 effectively prevents cancer cellization due to self-proliferation after completing the bone formation function.

Example 10 OB Cell Line Establishment

Embryonic stem cells (WA01 male embryonic stem cells, WiCell research institute) were aliquoted into 10 ⁶ cells in ⁶⁰ phi dishes, and the vector of Example 2 was then transfected. After 48 hours of transfection, cells without neoR gene were killed by treatment with neomycin for 5 days, and cells into which BMP-2 gene was introduced were selected.

Selected cells were differentiated into embryonic bodies (EB, Embryoid body), and then cultured for 3 weeks in bone differentiation medium containing adenosine (adenosine) to establish a cell line consisting of osteoblasts (OB, Osteoblast).

Thereafter, the plasmid DNA containing pCAG-Cre was transfected into the cell line so that the BMP-2 and HSV-tk genes were expressed by reacting the loxP gene with the Cre protein. 14 is an image of the osteoblasts (OB) differentiated by inserting the embryonic stem cells, empty vectors or the vector of Example 2 thereto.

BMP-2 expression of the cell line was confirmed by ELISA analysis, and the results are shown in FIG. 15. Referring to FIG. 15, it was confirmed that the OB cell line into which BMP-2 was introduced exhibited enhanced extracellular BMP-2 release compared to the control OB cell line into which the empty vector was introduced.

Example 11: Determination of Bone Formation Related Marker Expression Levels

To determine whether the OB cell line of Example 10 increases the expression of ALP, RUNX2, OSX, IBSP, SPP1 and OCN, which are related markers of bone formation, the gene expression level of each marker was measured in the same manner as in Example 6. It was. At this time, an OB cell line into which the empty vector was introduced was used as a control, and the measurement results are shown in FIG. 16.

Referring to FIG. 16, it can be seen that the OB cell line of Example 10 increases the expression of all of the bone formation markers ALP, RUNX2, OSX, IBSP, SPP1, and OCN. Suggests that it can effectively induce bone formation.

Example 12 Late Bone Formation Induction Evaluation

In order to evaluate the late bone formation ability of the OB cell line of Example 10, calcium and mineral deposition was measured in the same manner as in Example 7, and the results are shown in FIGS. 17 and 18, respectively.

Referring to FIGS. 17 and 18, the OB cell line of Example 10 exhibited two-fold improvement in calcium deposition and mineral deposition compared to the control group into which the empty vector was introduced, indicating that the late bone formation ability was excellent.

Example 13: Assessment of Bone Formation in Animal Models

In order to confirm the bone regeneration effect of the OB cell line of Example 10 in the skull-deficient animal model, bone formation was evaluated through the same H & E tissue staining test as in Example 8, and the results are shown in FIG. 19.

At this time, when no treatment was performed (Defect only), when only BCP support was applied (Scaffold), when BMP-2 injected from the outside was applied together with the support (50 ng / ml and 5 µg / ml), and the empty vector was The case where the introduced OB cell line was applied with the support was set as a control.

Referring to FIG. 19, it can be seen that an excellent bone formation effect can be realized when the OB cell line of Example 10 is applied as compared to the controls.

Example 14 Genome Editing of HGPRT Gene—Dual Kill Switch Implementation

In order to introduce a dual kill switch system by removing the HGPRT coding gene present on the X chromosome, the Cas9 / CRISPR method was performed. An sgRNA (guide RNA) complementary to the PAM site at the HGPRT gene exon8 site was cloned. Cas9 plasmid DNA and sgRNA were introduced into the TDF cell line of Example 3 by using a Neon Transfection method in a ratio of 1: 1 to prepare a double kill switch expression vector.

In order to confirm whether the HGPRT gene was removed, RNA was extracted from the cell line (double kill switch group) into which the double kill switch expression vector was introduced and the TDF cell line (control) of Example 3, and the extracted RNA was synthesized into cDNA. After that, the amount was measured using GAPDH, HGPRT real time Primer. The annealing temperature was 60, and the primers used were as follows.

Forward primer: TGACACTGGCAAAACAATGCA

Reverse primer: GGTCCTTTTCACCAGCAAGCT

Referring to FIG. 24, the HGPRT expression level of the double kill switch group was measured to be about 0.328 times that of the control group, and these results confirm that most of the HGPRT genes were removed.

Example 15: Securing Dual Kill Switch Cell Lines Using Drug Testing

The TDF cell line knocked out of the HGPRT gene in Example 14 and the TDF cell line of Example 3 were treated with 6-TG (Tioguanine), and the number of cells 5 and 9 days after the treatment was observed and is shown in FIG. 20. And surviving HGPRT gene knockout cells were isolated.

The isolated HGPRT gene knockout cell line and the TDF cell line of Example 3 were cultured in 6-well at 1 × 10 ⁴ . A 50 × aminopterins (HAT) stock in the culture was diluted with 1 × (hypoxanthine 100 μM, aminopretin 0.4 μM, thymidine 16 μM) and treated with aminopterin (HAT). In one culture, cell death after 48 hours of culture was observed and is shown in FIG. 21.

Referring to FIG. 20, 9 days after 6-TG treatment, more cells survived in the HGPRT knockout cell line of Example 14 compared to the TDF cell line of Example 3. In addition, referring to Figure 21, when treated with aminopterin, the TDF cell line of Example 3 almost no change in the number of cells, it can be seen that the cell death appeared in the isolated HGPRT knockout cell line.

Although the embodiments have been described by the limited embodiments and the drawings as described above, various modifications and variations are possible to those skilled in the art from the above description. For example, the techniques described may be performed in a different order than the described method, and / or the components described may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the following claims.

 <110> KOREA UNIVERSITY

<120> COMPOSITION FOR PREVENTING OR TREATING BONE DISEASE HAVING

         EXCELLENT BONE REGENERATION ABILITY

<130> FPC-2018-0109 / PCT

<150> KR 10-2017-0020967

<151> 2017-02-16

<150> KR 10-2018-0017137

<151> 2018-02-12

<160> 4

<170> KoPatentIn 3.0

<210> 1

<211> 345

<212> DNA

<213> Homo sapiens

<400> 1

caagccaaac acaaacagcg gaaacgcctt aagtccagct gtaagagaca ccctttgtac 60

gtggacttca gtgacgtggg gtggaatgac tggattgtgg ctcccccggg gtatcacgcc 120

ttttactgcc acggagaatg cccttttcct ctggctgatc atctgaactc cactaatcat 180

gccattgttc agacgttggt caactctgtt aactctaaga ttcctaaggc atgctgtgtc 240

ccgacagaac tcagtgctat ctcgatgctg taccttgacg agaatgaaaa ggttgtatta 300

aagaactatc aggacatggt tgtggagggt tgtgggtgtc gctag 345

<210> 2

<211> 593

<212> DNA

<213> Homo sapiens

<400> 2

aattccgccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag 60

gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga 120

gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg 180

ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt 240

gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca 300

ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc 360

agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 420

tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc 480

ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaacgtctag gccccccgaa 540

ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca acc 593

<210> 3

<211> 1131

<212> DNA

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atggcttcgt accccggcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 60

ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 120

cgcccggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc ccacgggatg 180

gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240

gtacccgagc cgatgactta ctggcgggtg ctgggggctt ccgagacaat cgcgaacatc 300

tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360

atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420

cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 480

ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcggta ccttatgggc 540

agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600

accaacatcg tgcttggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660

cagcgccccg gcgagcggct ggacctggct atgctggctg cgattcgccg cgtttacggg 720

ctacttgcca atacggtgcg gtatctgcag tgcggcgggt cgtggcggga ggactgggga 780

cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 840

cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc 900

aacggcgacc tgtataacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960

tccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1020

ctgcaactta cctccgggat ggtccagacc cacgtcacca cccccggctc cataccgacg 1080

atatgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg a 1131

<210> 4

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<212> DNA

<213> Artificial Sequence

<220>

<223> Lgk leading peptide binding BMP-2 gene

<400> 4

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tgtaagagac accctttgta cgtggacttc agtgacgtgg ggtggaatga ctggattgtg 120

gctcccccgg ggtatcacgc cttttactgc cacggagaat gcccttttcc tctggctgat 180

catctgaact ccactaatca tgccattgtt cagacgttgg tcaactctgt taactctaag 240

attcctaagg catgctgtgt cccgacagaa ctcagtgcta tctcgatgct gtaccttgac 300

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cgctag

Claims (9)

A stem cell or a cell differentiated from the stem cell into which a dual kill switch expression vector containing the BMP-2 encoding gene and the HSV-tk encoding gene and knocked out the HGPRT encoding gene is introduced is effective. It comprises as a component, a composition for preventing or treating bone diseases. The method of claim 1, The stem cells are embryonic stem cells (ESC, Embryonic stem cells) or mesenchymal stem cells (MSC, Mesenchymal stem cells), the composition for preventing or treating bone diseases. The method of claim 1, Cells differentiated from the stem cells are fibroblasts (osteoblasts) or osteoblasts (osteoblast), a composition for preventing or treating bone diseases. The method of claim 3, The fibroblasts are teratoma-derived fibroblasts (TDF, Teratoma-derived fibroblast), the composition for preventing or treating bone diseases. The method of claim 1, The bone disease is one or more selected from the group consisting of bone defects, osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, osteoplasia and osteomalacia, composition for preventing or treating bone diseases. The method of claim 1, Expression of one or more markers selected from the group consisting of Alkaline phosphatase (ALP), Integrin binding sialoprotein (IBSP), Run-related transcription factor 2 (RUNX2), OSX (Osterix), Secreted phosphoprotein 1 (SPP1) and Osteocalcin (OCN) Increasing, preventing or treating a bone disease composition. The method of claim 1, The composition further comprises a scaffold, a composition for preventing or treating bone diseases. The method of claim 7, wherein The support is made of polycaprolactone (PCL, Polycaprolactone) or biphasic calcium phosphate (BCP, Biphasic calcium phosphate), the composition for preventing or treating bone diseases. Preparing a vector comprising a BMP-2 coding gene and an HSV-tk coding gene; Knocking out the HGPRT encoding gene in the vector; And And introducing the vector into stem cells or cells differentiated from the stem cells.

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