WO2022262029A1 - Method for inducing and/or enhancing repair of cartilage damage on basis of protein factor encoded by chemically modified mrna - Google Patents

Abstract

A method for inducing and/or enhancing the repair of cartilage damage on the basis of a protein factor encoded by a chemically modified mRNA. According to the method, at least one protein factor in a TGF-β family is expressed by a chemically modified mRNA. The chemically modified mRNA encoding the TGF-β family is introduced by means of local injection, and single or multiple protein factors encoding TGF-β are produced by a body's own cells or transfected human marrow mesenchymal stem cells, so that the encoded protein factor achieves the aim of inducing and/or enhancing the repair of cartilage damage. Provided is a clinical gene therapy method for treating osteoarthritis.

Description

A method for inducing and/or enhancing cartilage damage repair based on chemically modifying mRNA-encoded protein factors technical field

The invention relates to the technical field of osteoarthritis, in particular to a method for inducing and/or enhancing cartilage damage repair based on chemically modifying mRNA-encoded protein factors.

Background technique

Osteoarthritis (OA) is a chronic joint disease characterized by degeneration and destruction of articular cartilage and bone hyperplasia. The degeneration of articular cartilage in osteoarthritis is arthritis caused by traumatic and degenerative osteoarthritis and rheumatoid bone. It is a serious medical problem, and it is the fourth most disabling disease in my country and the third most disabling disease in Europe and the United States.

Currently, the vast majority of treatments for osteoarthritis are medications to control pain and keep swelling down. But the cartilage continues to be destroyed, unable to change the disease process, and eventually joint replacement must be performed, which is a pain point in the clinical treatment of osteoarthritis.

It has been reported that shortly after the joint surface is damaged, chondrocytes adjacent to the damaged surface show a small amount of mitosis accompanied by increased synthesis of glycosaminoglycans and collagen, but the cartilage matrix does not increase significantly, unable to Efficiently repairs defects by itself, which is also a congenital defect of cartilage. Therefore, inducing and/or enhancing the repair of cartilage damage is the key to the etiological treatment of osteoarthritis. Mature chondrocytes have very limited repair capacity and lose terminal differentiation function, so human bone marrow mesenchymal stem cells (hMSCs) with differentiated chondrocytes have become the main therapeutic candidates for cartilage repair.

Transforming growth factor-β (Transforming growth factor beta, referred to as TGF-β) is a multifunctional protein that can affect the growth, differentiation, apoptosis and immune regulation of various cells. Transfection of hMSCs by the TGF-β family with viral vectors such as adenoviruses and retroviruses has been shown to induce chondrogenesis consistently while avoiding their loss of function due to terminal differentiation. However, the use of adenoviruses and retroviruses in clinical gene therapy has major safety concerns, such as the induction of severe allergic reactions and malignant tumors.

Therefore, it is urgent to study a safe and effective method for clinical gene therapy to induce and/or enhance the repair of cartilage damage.

Contents of the invention

The purpose of the present invention is to address the deficiencies in the prior art, and provide a method for inducing and/or enhancing cartilage damage repair based on chemically modifying mRNA-encoded protein factors.

The object of the present invention is achieved through the following technical solutions:

The present invention provides a method for inducing and/or enhancing cartilage damage repair based on chemically modified mRNA encoding protein factors, comprising the following steps:

Step 1, using a specific DNA template to synthesize mRNA encoding at least one protein factor in the TGF-β family;

Step 2, chemically modifying the mRNA encoding TGF-β family in vitro;

Step 3, introducing the chemically modified mRNA encoding the TGF-β family, and transfecting the body's own cells or human bone marrow mesenchymal stem cells to produce single or multiple protein factors encoding the TGF-β family.

In the above technical solution, in step 1, the DNA template contains specific 5' and 3' untranslated region DNA sequences, a signal peptide sequence and at least one mRNA sequence encoding a TGF-β family protein factor.

In the above technical scheme, the 5' untranslated region sequence is shown in SEQ ID NO: 1, the 3' untranslated region sequence is shown in SEQ ID NO: 2, and at least one TGF-β family protein factor is encoded The mRNA sequence is shown in SEQ ID NO:3.

In the above technical solution, the TGF-β family includes TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-6, BMP-7, BMP-10 and FGF-18 protein factors.

In the above technical solution, in Step 3, the chemically modified mRNA encoding TGF-β family is introduced by local injection.

Beneficial effects of the present invention:

A method of inducing and/or enhancing the repair of cartilage damage based on chemically modifying mRNA encoding protein factors of the present invention is to express at least one protein factor in the TGF-β family by chemically modifying mRNA, and introduce the code by local injection methods such as joint cavity Chemically modify mRNA of the TGF-β family, and produce single or multiple mRNAs that play a key role in cell differentiation, collagen synthesis and matrix deposition in cartilage tissue engineering by the body's own cells or transfection of human bone marrow mesenchymal stem cells (hMSCs) A protein factor encoding TGF-β, and then enabling the encoded protein factor to achieve the purpose of inducing and/or enhancing the repair of cartilage damage. The present invention has confirmed through experiments that the chemically modified mRNA encoding TGF-β can express the target protein in 293T cells. Chemical modification of mRNA can successfully induce chondrogenic differentiation of human MSC cells. More importantly, from the general observation, the repair effect of cartilage defect in each treatment group is significantly improved compared with the non-filling group, in which hMSC and (encoding TGF-β3 chemical The repair effect of the modified mRNA+hMSC) group is better than that treated only with TGF-β3, which can confirm that the chemically modified mRNA encoding TGF-β has the effect of inducing and/or enhancing the repair of cartilage damage. Therefore, the present invention provides a safe and effective clinical gene therapy method for the treatment of osteoarthritis, which is of great significance in the treatment of osteoarthritis and has broad application prospects.

Description of drawings

Fig. 1 is an effect diagram of the expression of chemically modified mRNA encoding TGF-β3 in 293T cells according to an embodiment of the present invention.

Fig. 2 is an effect diagram showing that chemically modified mRNA encoding TGF-β3 according to an embodiment of the present invention can promote the growth of human bone marrow mesenchymal stem cells in a 3D culture environment.

Fig. 3 is an effect diagram showing that chemically modified mRNA encoding TGF-β3 according to an embodiment of the present invention can successfully induce chondrogenic differentiation of human MSC cells.

Fig. 4 is a schematic diagram of the gross anatomical result of repairing the chemically modified mRNA+hMSC group encoding TGF-β3 after cartilage defect in rats according to the embodiment of the present invention.

detailed description

The present invention will be further elaborated below in combination with the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

A method for inducing and/or enhancing cartilage damage repair based on chemically modifying mRNA-encoded protein factors of the present embodiment, comprising the following steps:

Step 1. Synthesis of specific DNA template

1. Double-stranded DNA encoding TGF-β family protein factors:

Double-stranded DNA encoding TGF-β family protein factors: gblock, directly ordered from IDT Company of the United States, the DNA sequence (995bp) information is as follows:

(1) T7 promoter: TAATACGACTCACTATAGGG.

(2) 5' untranslated region sequence:

(3) TGF-β3 open reading frame sequence:

(4) 3' untranslated region sequence:

2. Add the poly-(a) tail DNA template of mRNA by PCR:

Prepare the PCR master mix according to Table 1 (the total volume is 200ul, and each of the eight reactions is 25ul)

Table 1. Composition of PCR Master Mix

components	Dosage	Final concentration
Kapa PRC mix(2X)	100uL	1X
Tail Primer-F1 10um	6uL	0.3uM
Tail Primer-T120 10um	6uL	0.3uM
water	80uL	the
gBlock DNA 10ug/ul	8uL	40-400pg/ul

Tailing primer -F1: 5'-TTGGACCCTCGTACAGAAGCTAATACG-3'.

Tail-F2-T120:

5 - TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCTACTCAGGCTTTATTCAAAGACCA-3.

3. Use the conditions in Table 2 to expand the PCR:

Table 2. PCR conditions

Cycles	transsexual	annealing	expand
1	95℃,2–3min	the	the
2-31	98℃,20s	60℃,15s	72℃,60s
32	72℃,3min	the	the

4. Check the quality of PCR products by gel electrophoresis.

5. Gel cutting and recovery of PCR products (QIAquick PCR purification kit, Qiagen, cat.no.28106):

The final concentration of the tail template was adjusted to 100ng/ul, which was used as a template for in vitro transcription and synthesis of mRNA.

Step 2. In vitro transcription and synthesis of mRNA

1. Assemble the mRNA cap structure and nucleotide mixture according to Table 3:

Cap structure 3′-O-Me-m7G(5′)ppp(5′)G RNA cap analog(New England Biolabs,cat.no.S1411S),-Methylcytidine-5′-triphosphate(Me-CTP; Trilink,cat .no.N1014), Pseudouridine-5'-triphosphate (Pseudo-UTP; Trilink, cat.no.N1019), other components are from MEGAscript T7 kit (Ambion, cat.no.AM1334).

Table 3. mRNA cap structure

2. Assemble the mRNA in vitro transcription system according to Table 4:

Table 3. mRNA in vitro transcription system

3. Place the reaction in a PCR instrument and incubate at 37°C for 3-6 hours.

4. Add 2ul Turbo DNase (from MEGAscript T7 kit, Ambion, cat.no.AM1334) to each sample.

5. Mix gently and incubate at 37°C for 15 minutes.

6. Use MEGAclear kit (Ambion, cat.no.AM1908) to purify the DNase-treated reaction; elute the modified mRNA with a total of 100 ul of elution buffer (50 ul of elution buffer is eluted twice).

7. Treat the purified modified mRNA with phosphatase (Antarctic phosphatase (New England Biolabs, cat. no. M0289S).

8. To each sample (~100ul), add 11ul 10X phosphatase buffer, then add 2ul phosphatase; mix the samples gently and incubate at 37°C for 0.5~1h.

9. After elution, measure the concentration of the modified mRNA in a NanoDrop spectrophotometer. The expected total yield should be ~50ug (30-70ug range; 100ul elution volume for a 40ul IVT reaction is 300-700ngul). Adjust the concentration to 100 ng/ul by adding elution buffer or TE buffer (pH 7.0).

Step 3: Transfect human bone marrow mesenchymal stem cells with chemically modified mRNA encoding TGF-β3

1. Rat bone and joint defect model

3-month-old athymic rats were anesthetized with intraperitoneal injection of pentobarbital, placed in a supine position, exposed the knee joint, made an incision on the upper midline of the patella, retracted the skin, and made a medial arthrotomy incision, making the patella toward The lateral displacement exposes the femoral trochlear of the knee joint, and creates a 1.5X1.5mm defect that cannot heal itself with a drill on the midline of the femoral trochlea.

2. In vitro transfection of chemically modified mRNA encoding TGF-β3

Primary human bone marrow mesenchymal stem cells, the number is 250,000, centrifuged in 0.5ml standard chondrogenic medium for 5 minutes under the condition of 2500 rpm, encoding TGF-β3 chemically modified mRNA, transfected with PEI, recessive The control group was chemically modified mRNA encoding GFP (GFP group), and the positive control group was TGF-β3 protein factor. Histological morphology and quantitative analysis (H&E, Safranin-O, Alcian Blue, Aggrecan, Collagen II staining) were performed after 14 days of culture.

3. Filling human bone marrow mesenchymal stem cells into a rat bone and joint defect model

After the human bone marrow mesenchymal stem cell microspheres were cultured in 3D in vitro for 14 days, they were filled in the bone and joint defects of rats. Morphology and CT observations were performed after filling, and they were divided into three groups, namely: no filling control group, encoding TGF- β3 chemically modified mRNA group (abbreviated as TGF-β3 group), encoding TGF-β3 chemically modified mRNA+human bone marrow mesenchymal stem cell microsphere group (abbreviated as TGF-β3 chemically modified mRNA+hMSC group).

Experimental results and analysis:

1. As shown in Figure 1, quantitative PCR, western blotting and immunofluorescence all confirmed that the chemically modified mRNA encoding TGF-β3 can express the target protein in 293T cells.

In Figure 1:

A: After the chemically modified mRNA encoding TGF-β3 was transfected into 293T cells, the cell protein was collected and detected by Western blot, which indicated that the chemically modified mRNA encoding TGF-β3 could be translated into TGF-β3, and the control group was the DNA plasmid encoding TGF-β3 carrier;

B: Real-time quantitative PCR shows that the translation efficiency of chemically modified mRNA encoding TGF-β3 is 5 times that of the control group;

C: Immunofluorescent staining of cells indicated that protein immunoblot detection and quantification showed that chemically modified mRNA encoding TGF-β3 was expressed in 293T cells, and the control group was the field of light microscope.

2. As shown in Figure 2, chemically modified mRNA encoding TGF-β3 can be successfully transfected into human bone marrow mesenchymal stem cell (hMSC) microspheres cultured in 3D in vitro, and both cmRNA-TGF-β3 and TGF-β3 proteins can The size of BMSCs microspheres was significantly increased.

In Figure 2:

A: chemically modified mRNA encoding green fluorescent protein (GFP) can be transfected into 3D cultured human bone marrow mesenchymal stem cells, in the figure cmRNA(-): untransfected chemically modified mRNA, BF: optical microscope field of view; cmRNA(+): transfected Stain chemically modified mRNA;

B: Quantitative GFP fluorescence analysis indicated that the green fluorescence of the transfected GFP group was significantly higher than that of the non-transfected group;

C: chemically modified mRNA encoding TGF-β3 can be transfected and promote the growth of human bone marrow mesenchymal stem cells in 3D culture, and compared with the non-transfected group, the increase effect is more obvious, cmRNA(-): chemically modified without transfection RNA, cmRNA(+): Transfection chemically modified mRNA, TGFb3 protein factor treatment as positive control;

D: Quantitative analysis suggests that chemically modified mRNA encoding TGF-β3 can significantly promote the growth of human bone marrow mesenchymal stem cells in 3D culture compared with the untreated group, which is similar to the effect of TGF-β3 protein factor treatment.

3. As shown in Figure 3, the results of HE staining, immunohistochemistry and sGAG content showed that chemically modified mRNA encoding TGF-β3 could successfully induce chondrogenic differentiation of human MSC cells, but the cartilage microspheres in the group treated with chemically modified mRNA encoding TGF-β3 Compared with the blank control group and positive control treatment group, the content of sGAG in the medium was significantly increased.

In Figure 3:

A: HE staining and chondrogenic immunohistochemical results show that chemically modified mRNA encoding TGF-β3 can successfully induce chondrogenic differentiation of human MSC cells, cmRNA(-): untransfected chemically modified RNA, cmRNA(+): transfected chemically Modified mRNA, TGF-β3 protein factor treatment as positive control;

B: The detection of the chondrogenic specific index sGAG content indicated that the ability of chemically modified mRNA encoding TGF-β3 to induce human MSC cells was significantly higher than that of the non-transfected chemically modified RNA group and the TGF-β3 protein factor treatment group.

4. As shown in Figure 4, general observations suggest that the repair effect of the cartilage defect in each treatment group is significantly improved compared with the non-filling group, but the repair effect of hMSC and chemically modified mRNA+hMSC encoding TGF-β3 is higher than that of the two groups. The treatment of TGF-β3 is better, and it can be confirmed that the chemically modified mRNA encoding TGF-β3 has the effect of inducing and/or enhancing the repair of cartilage damage.

From top to bottom in Figure 4:

The first group: rat cartilage defect was repaired for 2 weeks (2W), no treatment group (control) had no change in cartilage defect, collagen (collagen) + chemically modified mRNA group encoding TGF-β3, collagen (collagen) + rat bone marrow BMSCs group, collagen + chemically modified mRNA encoding TGF-β3 + rat bone marrow mesenchymal stem cells (BMSCs) group, all showed defect reduction and repair, but collagen + encoding TGF-β3 chemically modified Modified mRNA + rat bone marrow mesenchymal stem cells (BMSCs) group is the best;

The second group: cartilage defect was repaired for 4 weeks (4W), no treatment group (control) had no change in cartilage defect, collagen (collagen) + chemically modified mRNA group encoding TGF-β3, collagen (collagen) + rat bone marrow mesenchyme Stem cells (BMSCs) group, collagen (collagen) + encoding TGF-β3 chemically modified mRNA + rat bone marrow mesenchymal stem cells (BMSCs) group, all appeared to shrink and repair defects, but collagen (collagen) + encoding TGF-β3 chemically modified mRNA + Rat bone marrow mesenchymal stem cells (BMSCs) group is the best;

The third group: cartilage defect was repaired for 6 weeks (6W), no treatment group (control) had no change in cartilage defect, collagen (collagen) + chemically modified mRNA group encoding TGF-β3, collagen (collagen) + rat bone marrow mesenchyme Stem cells (BMSCs) group, collagen (collagen) + encoding TGF-β3 chemically modified mRNA + rat bone marrow mesenchymal stem cells (BMSCs) group, all appeared to shrink and repair defects, but collagen (collagen) + encoding TGF-β3 chemically modified mRNA + Rat bone marrow mesenchymal stem cells (BMSCs) group was the best. Simple collagen (collagen) treatment of defects did not change significantly.

The above examples are preferred implementations of the present invention, and are only used to illustrate the present invention conveniently, and are not intended to limit the present invention in any form. Anyone with ordinary knowledge in the technical field, if they do not depart from the present invention, Within the scope of the technical features, the equivalent embodiments that utilize the technical content disclosed in the present invention to make partial changes or modifications without departing from the technical features of the present invention still belong to the scope of the technical features of the present invention.

Claims (5)

1. A method for inducing and/or enhancing cartilage damage repair based on chemical modification of mRNA encoding protein factors, characterized in that: comprising the following steps:

   Step 1, using a specific DNA template to synthesize mRNA encoding at least one protein factor in the TGF-β family;

   Step 2, chemically modifying the mRNA encoding TGF-β family in vitro;

   Step 3, introducing the chemically modified mRNA encoding the TGF-β family, and transfecting the body's own cells or human bone marrow mesenchymal stem cells to produce single or multiple protein factors encoding the TGF-β family.
2. The method according to claim 1, characterized in that: in step one, the DNA template contains specific 5' and 3' untranslated region sequences, signal peptide sequences and at least one mRNA sequence encoding a TGF-β family protein factor .
3. The method according to claim 1, characterized in that: said 5' untranslated region sequence is as shown in SEQ ID NO:1, said 3' untranslated region sequence is as shown in SEQ ID NO:2, said at least The mRNA sequence encoding a TGF-beta family protein factor is shown in SEQ ID NO:3.
4. The method according to claim 1 or 2, characterized in that: the TGF-β family includes TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-6, BMP-7, BMP-10 and FGF-18 protein factor.
5. The method according to claim 1, characterized in that: in step 3, the chemically modified mRNA encoding TGF-β family is introduced by local injection.

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