Application of active substance in improvement of erythroid progenitor cell differentiation efficiency

Abstract

The invention relates to application of an active substance to improvement of erythroid progenitor cell differentiation efficiency, and belongs to the technical field of medicines. The active substance is selected from at least one of an Ataxin-2 inhibitor, an ATAD5 inhibitor, a histone-lysine N-methyltransferase inhibitor, a CREBBP inhibitor and a CBP ligand. Aiming at the problem of low efficiency of differentiation of hematopoietic stem / progenitor cells into erythrocytes in the conventional technology, screening is carried out by using massive compounds, adjustment and repeated screening are carried out according to a preliminary screening result, and after trial, a part of compounds are found to have the effect of improving the efficiency of differentiation of erythroid progenitor cells into erythroid cells; summary and conclusion are carried out according to the compounds, it is found that the target inhibitor has the effect of improving the efficiency of differentiating erythroid progenitor cells into erythroid cells, and the target inhibitor can be applied to the erythroid cell differentiation process to improve the differentiation efficiency.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of active substances in improving the differentiation efficiency of erythroid progenitor cells. Background Art

[0002] Red blood cells are a type of anucleated cell in the blood, also known as "erythrocytes" or "red blood cells" in blood cells. In the human body, red blood cells transport oxygen from the lungs to body tissues and cells, and take carbon dioxide back to the lungs to be excreted from the body. Because red blood cells have good biocompatibility, they can be used as a potential drug delivery carrier. And delivering drugs with red blood cells helps to improve the stability of drugs, increase the half-life of drugs in the body, and improve the therapeutic effect of drugs.

[0003] However, the application of red blood cells in drug delivery still faces some constraints, the most important of which is the production problem. At present, red blood cells and other blood products are mainly derived from peripheral blood donations from volunteers. Although the amount of blood donations is increasing, it is still in short supply.

[0004] In view of the above reasons, researchers have turned their attention to obtaining red blood cells through the in vitro erythropoiesis pathway, which means that red blood cells are produced by culturing cells in vitro without relying on the bone marrow and other tissues in the human body. The conventional in vitro erythropoiesis pathway is to differentiate human CD34+ hematopoietic stem / progenitor cells into red blood cells through in vitro induction methods and culture systems. However, the existing methods for differentiating CD34+ hematopoietic stem / progenitor cells into red blood cells cannot meet the needs.

[0005] Therefore, how to improve the efficiency of CD34+ hematopoietic stem / progenitor cells differentiating into erythrocytes has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] Based on this, it is necessary to provide an active substance for improving the differentiation efficiency of erythroid progenitor cells to address the problem of low efficiency of CD34+ hematopoietic stem / progenitor cells differentiating into erythroid cells, so as to improve the efficiency of producing erythroid cells in vitro.

[0007] The present invention discloses an application of an active substance in improving the differentiation efficiency of erythroid progenitor cells. The active substance is selected from at least one of an Ataxin-2 inhibitor, an ATAD5 inhibitor, a histone-lysine N-methyltransferase inhibitor, a CREBBP inhibitor, and a CBP ligand.

[0008] In view of the problem of low efficiency in the differentiation of hematopoietic stem / progenitor cells into erythroid cells in conventional technologies, a large number of compounds were screened and adjusted based on the initial screening results. After repeated screening and attempts, it was found that some compounds have the effect of improving the efficiency of erythroid progenitor cell differentiation into erythroid cells. Based on this type of compounds, it was found that the use of the above-mentioned target inhibitors has the effect of improving the efficiency of erythroid progenitor cell differentiation into erythroid cells. The above-mentioned target inhibitors can be used in the erythroid cell differentiation process to improve the differentiation efficiency.

[0009] In some embodiments, the active substance is selected from at least one of the following compounds:

[0010]

[0011]

[0012] In some embodiments, the erythroid progenitor cells differentiate into erythroid cells;

[0013] The erythroid cells are selected from erythroid colony forming units or colony forming unit erythrocytes (CFU-E), primitive erythrocytes, proerythrocytes, mesoerythrocytes, metaerythrocytes, reticulocytes and / or mature erythrocytes.

[0014] In some of these embodiments, the erythroid progenitor cells are selected from: umbilical cord blood stem cells, induced pluripotent stem cells (iPSCs), CD34+ cells, hematopoietic stem / progenitor cells, spleen colony forming (CFU-S) cells, myeloid progenitor cells (CMPs), blast colony forming cells, burst forming unit-erythroid cells / erythrocytes (BFU-E) and / or megakaryocyte-erythroid progenitor (MEP) cells.

[0015] In some embodiments, the hematopoietic stem cells, hematopoietic progenitor cells and / or hematopoietic stem / progenitor cells are CD34 positive cells.

[0016] In some of these embodiments, the active substance is an inhibitor against at least one of Ataxin-2, ATAD5, histone-lysine N-methyltransferase and CREBBP, and / or a CBP ligand;

[0017] The inhibitor produces an inhibitory effect at at least one of the following levels: gene expression level, mRNA transcription level, protein expression level and protein activity level. The ligand is a ligand that reduces CBP biotinylation.

[0018] In some of the embodiments, the inhibitor that produces an inhibitory effect at the gene expression level is selected from at least one of a CRISPR-Cas system reagent, a siRNA reagent, and an ASO reagent.

[0019] The invention also discloses the application of the active substance in preparing a promoter for improving the differentiation efficiency of erythroid progenitor cells.

[0020] The invention also discloses an erythroid cell differentiation promoter, comprising the above active substance.

[0021] In some embodiments, the erythroid differentiation promoter further comprises: erythroid differentiation complete medium. In some embodiments, the erythroid differentiation complete medium is supplemented with one or more of hydrocortisone, IL-3, SCF and EPO.

[0022] The present invention also discloses a method for improving the differentiation efficiency of erythroid progenitor cells, comprising the following steps: contacting the above active substance with erythroid progenitor cells.

[0023] In some embodiments, the method further comprises: introducing a nucleic acid into the erythroid progenitor cells and generating an engineered erythroid cell population.

[0024] In some embodiments, the nucleic acid encodes one or more exogenous polypeptides, and / or exogenous RNA.

[0025] In some of these embodiments, the engineered erythroid cell population comprises one or more exogenous proteins, and / or exogenous RNA.

[0026] In some embodiments, the method further comprises: click-conjugating one or more exogenous proteins to erythroid cells.

[0027] In some embodiments, the method further comprises: hypotonic loading of one or more exogenous proteins and / or exogenous RNA into erythroid cells.

[0028] In some embodiments, the method further comprises: loading one or more exogenous proteins and / or exogenous RNA into erythroid cells by physical manipulation.

[0029] In some of these embodiments, the one or more exogenous proteins, and / or exogenous RNA are present in the cytosol of the engineered erythroid cell population.

[0030] In some of these embodiments, the one or more exogenous proteins, and / or exogenous RNA are present on the membrane of the engineered erythroid cell population.

[0031] In some embodiments, the method comprises the following steps: dissolving the active substance, adding it to a complete erythroid differentiation medium, inoculating the erythroid progenitor cells, and performing differentiation culture to obtain erythroid cells.

[0032] In some embodiments, the concentration of the active substance in the differentiation culture system is 0.1-100 μM.

[0033] In some of the embodiments, the concentrations of hydrocortisone, IL-3, SCF and EPO in the differentiation culture system are 0.1-10 μM, 0.1-50 ng / ml, 10-500 ng / ml and 0.1-50 U / ml, respectively.

[0034] In some of the embodiments, during the differentiation culture process, the fluid is replenished every 2-4 days, and the cell density is adjusted according to the cell growth condition. The red blood cells are harvested after culturing for 15-20 days.

[0035] The invention also discloses a red blood cell differentiated by the above method.

[0036] The invention also discloses a vesicle produced by the red blood cells.

[0037] In some embodiments, the vesicles are secreted from the red blood cells or produced by extruding the cell membrane of the red blood cells.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention discloses an application of an active substance in improving the differentiation efficiency of erythroid progenitor cells. Such active substance can improve the efficiency of erythroid progenitor cells differentiating into erythroid cells, promote the engineering production efficiency of erythroid cells, and make the application prospect of erythroid cells in drug delivery broader. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The figure is the ratio of the number of cells in each group to the number of cells in the blank control group in Example 2;

[0041] Figure 2 It is a graph showing the ratio of the number of cells in each group in Example 2 to the number of cells in the solvent control group. DETAILED DESCRIPTION

[0042] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0044] The reagents used in the following examples, unless otherwise specified, are all commercially available; the methods used in the following examples, unless otherwise specified, are all achievable by conventional methods.

[0045] Example 1

[0046] In this example, human CD34 positive cells derived from mobilized peripheral blood were subjected to differentiation experiments into CFU-E to detect the effect of drugs on enhancing the differentiation of CD34+ HSC into erythroid cells.

[0047] The culture medium for CD34+HSC differentiation in this example is culture medium (purchased from STEMCELL), the test drugs (compounds 1-16) or DMSO in Table 1 were placed In the culture medium, the test drug was dissolved in DMSO. The final concentration of the medium was 10 μM, containing DMSO The volume percentage of DMSO in the culture medium was 0.01%, and no compound 1-16 or DMSO was added. The culture medium served as blank control.

[0048] Table 1. Test drugs (compounds 1-16)

[0049]

[0050]

[0051]

[0052]

[0053] CD34 positive cells were prepared and divided into 18 groups, namely blank control group, solvent control group and drug groups 1-16. The blank control group was treated with no addition of compound 1-16 or DMSO. The solvent control group used medium with only DMSO added. The cells in drug groups 1-16 were cultured with the addition of compounds 1-16. After counting CD34+HSCs with a cell counter, dilute the cells with IMDM 2% FBS to 10 times the final concentration of inoculation, and inoculate 1000 CD34+HSCs in each 35mm culture dish. Then add the diluted CD34+HSC suspension to the vortex vigorously immediately and then allow the air bubbles to dissipate. 1.1 mL of the mixture of culture medium and CD34+ HSC was added to each 35 mm culture dish. The dish was then placed in a CO 2 After 14 days of culture in the incubator, the CFU formation of each group was checked.

[0054] The experimental results of this example are as follows: after 14 days of differentiation culture, the number of CFU formed in drug group 3-16 is significantly greater than that in drug group 1-2, the blank control group and the solvent control group, indicating that compounds 3-16 can improve the efficiency of differentiation of erythroid progenitor cells into erythroid cells.

[0055] Example 2

[0056] In this example, human CD34 positive cells derived from mobilized peripheral blood were subjected to an experiment for differentiation into erythroid cells, to detect the effect of drugs on enhancing the differentiation of CD34+ HSC into erythroid cells.

[0057] The medium for CD34+ HSC differentiation in this example is Stempro 34 complete erythroid differentiation medium, which is prepared by the following method: 1 tube of 40 mL of StemPro-34 nutrient supplement (manufacturer GIBCO, catalog number 10641-025) is added to StemPro-34 SFM (manufacturer GIBCO, catalog number 2373885) and mixed, and then GlutaMAX TM -I (100X) supplement (manufacturer: Thermo, catalog number 25030) was added to make the final concentration 1X, and then hydrocortisone, IL-3, SCF and EPO were added to make their working concentrations in Stempro 34 erythroid differentiation complete medium to be 1 μM, 5 ng / mL, 100 ng / mL and 3 U / mL, respectively.

[0058] In this example, the test drugs (compounds 1-16) or DMSO in Table 1 were placed in Stempro 34 complete medium, the test drugs were dissolved in DMSO, the final concentration of the test drugs in Stempro 34 erythroid differentiation complete medium was 10 μM, the volume percentage of DMSO in the Stempro 34 erythroid differentiation complete medium containing DMSO was 0.01%, and Stempro 34 erythroid differentiation complete medium without adding compounds 1-16 or DMSO was used as a blank control.

[0059] Human CD34 positive cells were used for cell inoculation, and the cells were divided into 18 groups, namely blank control group, solvent control group and drug groups 1-16. The blank control group was cultured with Stempro 34 erythroid differentiation complete medium without adding compound 1-16 or DMSO, the solvent control group was cultured with Stempro 34 erythroid differentiation complete medium with only DMSO added, and drug groups 1-16 were cultured with Stempro 34 erythroid differentiation complete medium with compound 1-16 added. Among them, the blank control group and the solvent control group were 4 replicates, and the drug groups 1-16 were 2 replicates. During the differentiation culture process, the fluid was replenished every 2-4 days, and the cell density was adjusted according to the cell growth. After 18 days of culture, the cell clusters were visible red, and the red blood cells were harvested and counted.

[0060] The RBC yield of each group is shown in Table 2 below. Figure 1 and Figure 2 They are the ratio graphs of various cell numbers to the cell number of the blank control group, and the ratio graphs of various cell numbers to the cell number of the solvent control group.

[0061] Table 2. RBC production in each group (×10 5 cells)

[0062] Group Duplicate hole 1 Duplicate hole 2 Duplicate hole 3 Duplicate hole 4 average value Blank control group 3.45 3.69 3.55 2.98 3.4175 Solvent control group 3.3 3.05 2.88 3.45 3.17 Drug Group 1 2.33 2.43 / / 2.38 Drug Group 2 1.48 1.6 / / 1.54 Drug Group 3 8 6.37 / / 7.185 Drug Group 4 3.98 4.65 / / 4.315 Drug Group 5 4.21 4.67 / / 4.44 Drug Group 6 8.52 7.7 / / 8.11 Drug Group 7 6.16 6.52 / / 6.34 Drug Group 8 4.2 4.57 / / 4.385 Drug Group 9 3.74 4.1 / / 3.92 Drug Group 10 2.92 4.93 / / 3.925 Drug Group 11 4.29 4.85 / / 4.57 Drug Group 12 4.05 4.1 / / 4.075 Drug Group 13 5.61 5.36 / / 5.485 Drug Group 14 3.49 4.67 / / 4.08 Drug Group 15 9.76 9.3 / / 9.53 Drug Group 16 4.52 5.1 / / 4.81

[0063] The results showed that compounds 3-16 were able to improve the efficiency of erythroid progenitor cells differentiating into erythroid cells. Compounds 3, 6 and 15 significantly improved the efficiency of erythroid progenitor cells differentiating into erythroid cells, and the RBC production was more than twice that of the blank control group.

[0064] Further analysis of the above compounds revealed that some of the compounds have certain commonalities, such as being inhibitors of certain targets (see the table below for details), suggesting that such target inhibitors have the same function as the present invention in promoting the differentiation of erythroid progenitor cells into erythroid cells.

[0065] Table 3 Analysis of each compound

[0066]

[0067]

[0068] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0069] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of an active substance in improving the differentiation efficiency of erythroid progenitor cells, characterized in that: The active substance is selected from at least one of: an Ataxin-2 inhibitor, an ATAD5 inhibitor, a histone-lysine N-methyltransferase inhibitor, a CREBBP inhibitor, and a CBP ligand.

2. The use according to claim 1, characterized in that: The active substance is selected from at least one of the following compounds:

3. The use according to claim 1, characterized in that: The erythroid progenitor cells differentiate into erythroid cells; The erythroid cells are selected from erythroid colony-forming units or colony-forming unit erythrocytes, primitive erythrocytes, proerythrocytes, mesoerythrocytes, metaerythrocytes, reticulocytes and / or mature erythrocytes.

4. The use according to claim 1, characterized in that: The erythroid progenitor cells are selected from: umbilical cord blood stem cells, induced pluripotent stem cells, CD34+ cells, hematopoietic stem / progenitor cells, spleen colony-forming cells, myeloid progenitor cells, blast colony-forming cells, burst-forming unit-erythroid cells / erythrocytes and / or megakaryocyte-erythroid progenitor cells.

5. The use according to claim 4, characterized in that: The hematopoietic stem / progenitor cells are CD34 positive cells.

6. The use according to any one of claims 1 to 5, characterized in that: The active substance is an inhibitor against at least one of Ataxin-2, ATAD5, histone-lysine N-methyltransferase and CREBBP, and / or a CBP ligand; The inhibitor produces an inhibitory effect at at least one of the following levels: gene expression level, mRNA transcription level, protein expression level and protein activity level. The ligand is a ligand that reduces CBP biotinylation.

7. The use according to claim 6, characterized in that: The inhibitor that produces an inhibitory effect at the gene expression level is selected from: at least one of a CRISPR-Cas system reagent, a siRNA reagent, and an ASO reagent.

8. Use of the active substance according to claim 1 in the preparation of a promoter for improving the differentiation efficiency of erythroid progenitor cells.

9. An agent for promoting erythroid cell differentiation, characterized in that: The invention comprises the active substance described in any one of claims 1 to 7.

10. The erythrocyte differentiation promoting agent according to claim 9, characterized in that Also includes: Complete erythroid differentiation medium.

11. The erythrocyte differentiation promoting agent according to claim 10, characterized in that The complete erythroid differentiation medium is supplemented with one or more of hydrocortisone, IL-3, SCF and EPO.

12. A method for improving the differentiation efficiency of erythroid progenitor cells, characterized in that: The following steps are involved: The active substance according to any one of claims 9 to 11 is brought into contact with erythroid progenitor cells.

13. The method according to claim 12, characterized in that The method further comprises: Nucleic acids are introduced into the erythroid progenitor cells, and a population of engineered erythroid cells is generated.

14. The method according to claim 13, characterized in that The nucleic acid encodes one or more exogenous polypeptides, and / or exogenous RNA.

15. The method according to claim 13, characterized in that The engineered erythroid cell population comprises one or more exogenous proteins, and / or exogenous RNA.

16. The method according to claim 12, characterized in that The following steps are involved: The active substance is dissolved and added into a complete erythroid differentiation medium, and the erythroid progenitor cells are inoculated and differentiated to obtain erythroid cells.

17. The method according to claim 16, characterized in that: The concentration of the active substance in the differentiation culture system is 0.1-100 μM.

18. The method according to claim 16, characterized in that During the differentiation culture process, the fluid is replenished every 2-4 days, and the cell density is adjusted according to the cell growth situation. The erythroid cells are harvested after culturing for 15-20 days.

19. An erythroid cell, characterized in that: Differentiated by the method according to any one of claims 12 to 17.

20. A vesicle, characterized in that Produced by the erythroid cells of claim 18.

21. The vesicle according to claim 19, characterized in that It is produced by secretion of the erythroid cells and / or by extrusion of the cell membrane of the erythroid cells.

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