KR20210116173A - Recombinant lentivirus overexpressing cartilage regeneration signal cell growth factor and pharmaceutical composition for cartilage regeneration including it - Google Patents

Abstract

The present invention relates to a method for manufacturing a cell line overexpressing a cartilage regeneration signal cell growth factor using adipose tissue-derived stem cells and a mixture of cell line cultures manufactured by using the same. Cell lines overexpressing a specific signal substance can standardly manufacture high-concentration mixed CM without changing the content of secretions other than a target substance, and mixed CM can be used as a cartilage regeneration treatment for chondrocyte activation.

Description

Recombinant lentivirus overexpressing cartilage regeneration signal cell growth factor and cartilage regeneration pharmaceutical composition comprising the same

The present invention relates to a recombinant lentivirus overexpressing a cartilage regeneration signal cell growth factor and a pharmaceutical composition for cartilage regeneration comprising the same, and more particularly, to a cell growth known as a cartilage regeneration signal by introducing a gene into an adipose tissue-derived adult stem cell. It relates to a method for producing a cell line having a pharmaceutical effect by establishing cell lines overexpressing a factor, and to a mixture of conditioned media (CM) produced thereby.

Changes in the biomechanical properties of cartilage and chondrocytes of joints or intervertebral discs due to aging or injury lead to deterioration of the tissue matrix, causing severe pain and disability. Because of the limited internal regenerative capacity of cartilage, surgery and conservative treatments are used as methods for tissue recovery and pain relief. However, since the long-term outcome of the disease is not good with current treatment methods, effective treatment methods for osteoarthritis are still unsatisfactory despite their widespread use.

Tissue engineering has been proposed as a promising method for cartilage treatment, the goal of which is to restore function by replacing a patient's cartilage defect with new cartilage formed in vitro. Various cellular sources, including stem cells and primary cells, natural or synthetic scaffold materials, signaling molecules, and mechanical stimuli, have been studied to improve the biological and biomechanical functions of engineered cartilage.

Nevertheless, the clinical application of engineered cartilage faces difficulties.

That is, when engineered cartilage is transplanted without properly reproducing the characteristics of the native tissue, the disadvantages of unstable phenotype and poor fusion with surrounding native tissue are still major challenges to be solved.

Many recent studies have reported that treatment using stem cells in degenerative diseases has a beneficial effect, and stem cells have improved tissue repair due to their ability to secrete signaling molecules that have a beneficial effect on damaged tissues rather than their ability to differentiate into necessary tissues. It is said to cause

Signaling molecules transmit signals between cells and play an important role in the replenishment of extracellular matrix materials following cell activation and related tissue repair. Among them, the transforming growth factor-β (TGFβ) subfamily, bone morphogenetic proteins (BMPs), insulin-like growth factors (IGFs), and fibroblast growth factors (FGFs) are major growth factors related to cartilage development and homeostasis. It has been reported and provides important clues for cartilage regeneration treatment.

In addition, from the study of various secretory factors derived from stem cells, it was shown that secreted factors without stem cells themselves can regenerate tissues under various conditions related to tissue and organ damage. These secreted factors are referred to as secretomes, microvesicles or exosomes and can be found in the medium in which stem cells are cultured. Therefore, this medium is called conditioned-media (CM) (HO Kim and S. Choi. Medicine, vol. 10, no. 3, pp. 93-01, 2013).

Using the secretome included in CM has several advantages. Compared to using stem cells, CMs have promising prospects as pharmaceuticals in regenerative medicine, as they are easier to manufacture, freeze-dried, packaged, and transported, and because they are cell-free, they can avoid the problem of immune rejection even when donors and recipients do not match. have.

However, the dose level for signal substances related to cartilage regeneration to show the regenerative therapeutic effect is much higher than the level contained in CM, so in laboratory conditions, it is necessary to concentrate the CM or purchase the substance in powder form and apply it after preparing it in high concentration. still have

(Thesis) H. O. Kim and S. Choi. Medicine, vol. 10, no. 3, pp. 93-01, 2013

The present invention has been devised to solve the above problems, and introduces genes into adipose tissue-derived mesodermal adult stem cells to develop cell lines that overexpress major signaling substances related to cartilage regeneration by 100 times or more, thereby solving the problem of effective concentration The purpose is to provide effective CM for cartilage regeneration by mixing each cell line and CM.

To achieve the above object, the present invention provides a recombinant lentiviral vector comprising at least one selected from the group consisting of genes encoding human-derived TGF-β1, BMP2, bFGF and IGF1 proteins.

In addition, the recombinant lentiviral vector is characterized in that it comprises one or more promoters.

In addition, the promoter is characterized in that at least one selected from the group consisting of cytomegalovirus (CMV), human elongation factor-1 alpha (EF-1α) and inducible promoter.

In addition, the vector is characterized in that it comprises an internal ribosome entry site (IRES).

Further, in order to achieve the above object, the present invention provides a recombinant lentivirus obtained by isolating the lentivirus from the transformed host cell after transforming the host cell with the recombinant lentiviral vector.

In addition, in order to achieve the above object, the present invention provides a recombinant lentivirus comprising at least one selected from the group consisting of genes encoding human-derived TGF-β1, BMP2, bFGF and IGF1 proteins.

Further, in order to achieve the above object, the present invention provides a host cell transfected with the recombinant lentivirus.

In addition, the host cell is characterized in that the mesenchymal stem cells including human adipose stem cells.

In addition, the present invention provides a pharmaceutical composition for cartilage regeneration comprising the recombinant lentivirus as an active ingredient to achieve the above object.

In addition, the present invention provides a pharmaceutical composition for cartilage regeneration comprising the host cell as an active ingredient in order to achieve the above object.

In addition, the present invention provides a pharmaceutical composition for cartilage regeneration using the culture (CM) of the host cell as an active ingredient to achieve the above object.

According to the present invention, a high concentration of mixed CM can be prepared standardly without change in the content of secretions other than the target substance produced by cell lines overexpressing a specific signal substance, and the mixed CM is used for cartilage regeneration for chondrocyte activation. It can be used as a therapeutic agent.

1 is a graph comparing the cell proliferation rate of immortalized human adipose stem cells and non-immortalized human adipose stem cells.
FIG. 2 is a schematic diagram of the genes inserted into the pLenti-TGF-β1-Puro, pLenti-BMP2-Puro, pLenti-bFGF-Puro, and pLenti-IGF1-Puro lentiviral vectors.
3 is an image comparing the morphologies of ASC cells before gene introduction and VAS1, VAS1-TGF-β1, pLenti-BMP2-Puro, VAS1-bFGF, and VAS1-IGF1 cell lines.
4 is an image comparing the contents of secreted cytokine/chemokine in ASC cells before gene introduction, and in VAS1, VAS1-TGF-β1, pLenti-BMP2-Puro, VAS1-bFGF, and VAS1-IGF1 cell lines.
5 is an experimental result showing the expression of transgenes in VAS1-TGF-β1, pLenti-BMP2-Puro, VAS1-bFGF, and VAS1-IGF1.
6 is a graph showing the cell numbers of VAS1-TGF-β1, pLenti-BMP2-Puro, VAS1-bFGF, and VAS1-IGF1 cells obtained by subculture.
7 is a table showing the content of signal substances in the CM of ASC, VAS1-TGF-β1, pLenti-BMP2-Puro, VAS1-bFGF, and VAS1-IGF1.
8 is a graph showing changes in the content of GAG and total collagen secreted by chondrocytes treated with various CMs.

The present invention relates to a recombinant lentiviral vector comprising at least one selected from the group consisting of genes encoding human-derived TGF-β1, BMP2, bFGF and IGF1 proteins.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

In one embodiment of the present invention, transforming growth factor TGF-β1, bone morphogenetic protein BMP2, insulin-like growth factor IGF1, and fibroblast growth factor bFGF protein are used as major growth factors for cartilage regeneration.

The lentiviral vector used in the present invention can be inserted into the genomic DNA of a cell to be infected to stably express and deliver the gene.

The recombinant lentiviral vector of the present invention may include cytomegalovirus (CMV), human elongation factor-1 alpha (EF-1α) or an inducible promoter.

In addition, a specific polynucleotide can be translated into a protein and function by being linked to another polynucleotide so that it can exert its function.

When two or more genes are transcribed from one promoter in the lentiviral vector of the present invention, an internal ribosome entry site (IRES) may be included so that each protein can be expressed from the single transcript. IRES can be used to produce a protein that performs a plurality of different functions with one promoter.

The present invention provides a recombinant lentivirus comprising genes encoding TGF-β1, bone morphogenetic protein BMP2, insulin-like growth factor IGF1, and fibroblast growth factor bFGF protein. A transformable lentivirus can be obtained by transforming a packaging cell, which is a host cell, and isolating the lentivirus from the packaging cell.

In addition, the present invention provides a host cell transfected with the recombinant lentivirus. "Transfection" means transferring a gene loaded into a recombinant lentiviral vector through viral infection.

In addition, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

[Example]

[Example 1. Preparation of immortalized human adipose stem cells (ASC)]

1-1. Preparation of Lentiviral Vector Containing Immortalization Gene

In order to immortalize ASC, a lentiviral vector containing immortalization genes v-myc and L-myc, respectively, was prepared.

First, the pLenti-Neo vector was constructed by removing the puromycin selection gene from the pLVX vector (Takara, USA) and substituting the Neomycin selection gene.

The v-myc gene was inserted into the pLenti-Neo lentiviral vector so that expression could be regulated by the CMV promoter. The constructed vector was named pLenti-v-myc-Neo.

On the other hand, the L-myc gene was inserted into the pLenti-Neo lentiviral vector so that expression can be regulated by the CMV promoter. The constructed vector was named pLenti-L-myc-Neo.

1-2. Production of Lentiviruses Containing Immortalization Genes

Using the lentiviral vector prepared in Example 1-1, a lentivirus including an immortalized gene was produced by the following method.

First, 293T cells, which are lenti-X packaged cells (Takara, USA), were cultured in a 150 mm dish using DMEM medium containing 10% fetal bovine serum (FBS). Meanwhile, lentiviral vectors were extracted and quantified from DH5α E. coli cells using the EndoFree Plasmin Maxi Kit (Qiagen, USA).

After washing the cultured lenti-X cells with PBS, 3 ml of TrypLE™ Select CTS™ (Gibco, USA) was added. After leaving the cells at 37° C. for about 5 minutes, it was confirmed that the cells were detached. The detached cells were neutralized by adding 7 ml of DMEM medium containing 10% FBS. The neutralized cells were collected in a 50 ml tube and centrifuged at 1,500 rpm for 5 minutes. The supernatant was removed and the cells were resuspended by adding 10 ml of DMEM culture medium containing 10% FBS. Suspended cells were counted with a hematocyte cytometer, and then ^{aliquoted to 1.2×10 7} cells in a 150 mm dish. When the seeded cells were cultured to a cell confluency of about 90%, 12 μg of lentiviral vector, 12 μg of psPAX (Addgene; gag-pol expression, packaging plasmid) and 2.4 μg of pMD.G plasmid (Addgene; VSVG) expression, envelope plasmid) mixture was transduced into the cells. To aid in transduction, lipofectamine (Invitrogen, USA) and plus agent (Invitrogen, USA) were used. After 6 hours of transduction, the medium was exchanged with DMEM containing 10% FBS. After further culturing for 48 hours, the supernatant was collected.

The obtained supernatant was mixed with a lentivirus concentration kit (Lenti-X concentrator, Takara, USA), and then incubated at 4°C overnight. The virus was obtained by centrifugation at 4° C. and 4,000 rpm for 2 hours, which was resuspended in 0.5 ml of DMEM without FBS. As a result, the lentivirus produced from the lentiviral vector was used at a concentration of ^{1.0x10 8 MOI.}

1-3. Preparation of immortalized human adipose stem cells

Immortalized human adipose stem cells were prepared using the lentivirus containing the immortalized gene produced in Example 1-2.

First, human adipose stem cells were prepared as follows. Specifically, fats from healthy donors were obtained. This was mixed with 0.5% collagenase I in a sterile container to decompose the fat. After reacting the fat mixture at 37° C. for 1 hour, the supernatant was removed by centrifugation, washed with phosphate buffered saline (PBS), and a cell pellet was obtained. The obtained pellet was suspended in DMEM-low glucose (11885-084, Gibco, USA) medium containing 20% FBS and 5 ng/ml b-FGF (100-18B, Peprotech, USA) and dispensed into culture flasks. . This was cultured at 37° C., 5% CO ₂ conditions for 24 to 48 hours, and then replaced with a fresh medium. They were subcultured while replacing them with a new medium at intervals of 3 to 4 days, and after 2 weeks of culture, it was confirmed whether they were adipose stem cells using a fluorescence cytometer.

The selected cells were infected with pLenti-v-myc-Neo lentivirus at an MOI of 100. After infection, cells infected with pLenti-v-myc-Neo lentivirus were selected by adding 1 μg/ml of neomycin to the stabilized cell culture medium. The selected cells were designated as VAS1.

As a result, the cell proliferation rates of human adipose stem cells containing the immortalized gene and human adipose stem cells not containing the immortalized gene are shown in FIG. 1 . Human adipose stem cells (VAS1) infected with a lentivirus containing immortalized genes v-myc and c-myc maintained a high cell proliferation rate even after 12 passages of culture. On the other hand, normal human adipose stem cells (ASC) rapidly decreased the cell proliferation rate after 10 passages of culture.

[Example 2. Production of Lentivirus Containing Functional Gene]

2-1. Construction of Lentiviral Vector Containing TGF-β1 Gene

In addition, by inserting the IRES gene into the pLentiX lentiviral vector, it was designed to insert two genes. The puromycin resistance gene was inserted in the back of the IRES and the TGF-β1 gene was inserted in the front so that the expression of both genes could be regulated by the CMV promoter. The IRES is a ribosome binding site, allowing translation to start even without a 5'-cap structure, so that two proteins can be expressed in one mRNA. The constructed vector was named pLenti-TGF-β1-Puro, and a vector into which BMP2, bFGF, and IGF1 genes were inserted was prepared in the same manner. Vectors into which each gene was inserted were named pLenti-BMP2-Puro, pLenti-bFGF-Puro, and pLenti-IGF1-Puro, respectively, and the structures thereof are shown in FIG. 2 .

2-2. Production of Lentiviruses Containing Functional Genes

Using the pLenti-TGF-β1-Puro, pLenti-BMP2-Puro, pLenti-bFGF-Puro, and pLenti-IGF1-Puro lentiviral vectors constructed in Example 2-1, as described in Example 1-2, Lentiviruses were produced in the same way. The produced lentivirus was used at a concentration ^{of 1x10 8 MOI.}

[Example 3. Preparation of lentivirus-transfected human adipose stem cells containing functional genes]

3-1. Preparation of lentivirus-transfected human adipose stem cells containing functional genes

The immortalized human adipose stem cells prepared in Example 1-3 were infected with the lentivirus containing the functional gene produced in Example 2-2 into different VAS1 cells to express each functional gene. cells were prepared. Infection was performed in the same manner as described in Examples 1-3. After infection, cells infected with pLenti-TGF-β1-Puro, pLenti-BMP2-Puro, pLenti-bFGF-Puro, and pLenti-IGF1-Puro lentiviruses by adding 1 μg/ml of puromycin to the culture medium of the stabilized cells, respectively. was selected.

The selected cells were cultured to form colonies. Cell lines are established by culturing monoclonal cells from the formed colonies. Cells expressing TGF-β1 are VAS1-TGF-β1, cells expressing BMP2 are VAS1-BMP2, and cells expressing bFGF are VAS1-bFGF, IGF1. The expressing cells were named VAS1-IGF1.

3-2. Confirmation of surface antigen protein expression in established cell lines

The morphology of human adipose stem cells before gene insertion and the cell line into which the functional gene was introduced was almost unchanged from that of the existing VAS1, and the cell picture is shown in FIG. 3 . In addition, the expression of secreted cytokine/chemokine protein in cell lines introduced with functional genes was compared and analyzed with human adipose stem cells (ASC) and VAS1. The experiment was performed using the Cytokine profiling kit (R&D Systems, USA) using the manufacturer's method as it is. The experimental results are shown in FIG. 4 .

As shown in FIG. 4 , the content of cytokine/chemokine, which is a unique secreted protein possessed by adipose stem cells, is hardly changed in the cell line introduced with the functional gene, even after genetic manipulation to express the functional gene, and it is similar to that of human adipose stem cells The same was confirmed.

3-3. Confirmation of transgene expression of functional gene-introduced cell lines

Total RNA was isolated from the established functional gene-introduced cell line using the RNeasy mini kit (Qiagen, Germany). 50 ng of cDNA was used as a sample, and 100 ng of VAS1-TGF-β1 plasmid DNA as a positive control and 1 μl of dW as a negative control were added and PCR was performed to confirm the expression of the transgene, respectively.

A 1% agarose gel was placed in the electrophoresis kit. From the first well, 10 μl of each was loaded in the order of negative control, VAS1, and functional transgenic cell line samples (3 pieces). After that, electrophoresis was performed at 100 V for 20 minutes, and a photograph of the gel was taken and the results are shown in FIG. 5 .

As shown in FIG. 5 , PCR products were identified only in samples of functional transgenic cell lines.

3-4. Cell PDL (Population doubling level) analysis

The cell line into which the functional gene was introduced was seeded at a number ^{of 4x10 4 cells.} Cells were obtained by subculture for about 4 days, and the total number of cells was measured. The same number of cells was seeded and the number of cells was measured using a cell counter every 4 days, and the results are shown in FIG. 6 . As shown in FIG. 6 , it was confirmed that the long-term subculture showed stable growth.

[Example 4. CM acquisition of overexpressed cell lines (VAS1-TGF-β1, VAS1-BMP2, VAS1-bFGF, VAS1-IGF1) and measurement of cartilage regeneration effect of the mixture]

4-1. Cultivation of cell lines, CM harvesting, and quantification of signal substances

The cell lines obtained in Example 3 and the non-transduced stem cells (ASCs) were inoculated using a serum-free medium (StemPro MSC SFM CTS; Gibco) as many as 1.2 × 10 ^{7 in a 150 cm 2 flask coated with an adhesion protein, respectively.} did. CM was obtained after proliferating the cells in an incubator (37° C., 5% CO _{2 ) until 70-80% confluent.}

Signal substances in each obtained CM were quantified using enzyme-linked immunosorbent (ELISA) (Quantikine ELISA Kit, R&D system) (FIG. 7).

The overexpressed cell lines secrete each signal material about 100 times.

4-2. Measurement of cartilage regeneration effect of a mixture of four CMs

In order to examine the cartilage regeneration effect of the CM mixture obtained from each cell line, chondrocytes were cultured to measure changes in the contents of GAG (glycosaminoglycan) and total collagen, an extracellular matrix material of cartilage tissue.

^{That is, chondrocytes (NHAC-Kn-Human Knee Articular Chondrocytes; LONZA) were introduced into 6} wells with sterilized molten 2% agarose in a well-equipped state with polysulfone die, and 5 × 10 6 cells per well were inoculated for 10 days. Cell aggregates were obtained by culturing using a culture medium (CGM chodrocyte Growth Medium Bulletit). Then, the cell aggregates were transferred to 48 wells coated with 2% agarose gel, and each CM and mixed CM (concentration of each signal material = about 30 ng/ml) were treated with the culture medium for 4 weeks (CM is only in the first and third weeks) Daily treatment) was used for quantification of GAG and total collagen in cell aggregates obtained by culturing (Blyscan Glycosaminoglycan Assay kit (Biocolor); Collagen Assay kit (R%&D)).

8 is a diagram showing changes in the measured GAG and total collagen content. In the case of GAG, the group treated with single CM showed an average increase of about 50% compared to the control group, and the group treated with the mixture showed an average increase of 60%. In the case of collagen, the single CM treatment group increased by 8% compared to the control group, and the mixture treatment group showed an average increase of about 50%. Therefore, the CM mixture showed excellent effect in regenerating cartilage tissue by activating chondrocytes.

Claims (11)

A recombinant lentiviral vector comprising at least one selected from the group consisting of genes encoding human-derived TGF-β1, BMP2, bFGF and IGF1 proteins.
According to claim 1,
The recombinant lentiviral vector is a recombinant lentiviral vector, characterized in that it comprises one or more promoters.
3. The method of claim 2,
The promoter is a recombinant lentiviral vector, characterized in that at least one selected from the group consisting of cytomegalovirus (CMV), human elongation factor-1 alpha (EF-1α) and inducible promoter.
4. The method of claim 3,
Recombinant lentiviral vector, characterized in that the vector comprises an internal ribosome entry site (IRES).
A recombinant lentivirus obtained by isolating the lentivirus from the transformed host cell after transforming the host cell with the recombinant lentiviral vector of any one of claims 1 to 4.
A recombinant lentivirus comprising at least one selected from the group consisting of genes encoding human-derived TGF-β1, BMP2, bFGF and IGF1 proteins.
A host cell transfected with the recombinant lentivirus of claim 6.
8. The method of claim 7,
The host cell is a host cell, characterized in that the mesenchymal stem cells including human adipose stem cells.
A pharmaceutical composition for cartilage regeneration comprising the recombinant lentivirus of claim 6 as an active ingredient.
A pharmaceutical composition for cartilage regeneration comprising the host cell of claim 7 or 8 as an active ingredient.
A pharmaceutical composition for cartilage regeneration comprising the culture (CM) of the host cell of claim 7 or 8 as an active ingredient.

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