Application of RBM3 in preparation of preparation for treating thalassemia

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of RBM3 in preparation of a preparation for treating thalassemia, the nucleotide sequence of the RBM3 is shown as SEQ ID NO.4, and the amino acid sequence of the RBM3 is shown as SEQ ID NO.5. The invention discovers that down-regulation of RBM3 gene expression can significantly activate expression of gamma-globin for the first time, and the RBM3-related shRNA has a positive effect in preparation of drugs for treating beta-thalassemia. Meanwhile, in the experiment process, it is not found that bone marrow suppression exists in RBM3, the erythroid differentiation potential of hematopoietic stem cells is damaged, and the adverse effect of liver and kidney injury does not exist.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of RBM3 in the preparation of a preparation for treating thalassemia. Background Art

[0002] Thalassemia, also known as thalassemia or thalassemia, is a group of inherited hemolytic anemias classified primarily as α-thalassemia, β-thalassemia, δβ-thalassemia, and γδβ-thalassemia. β-thalassemia, also known as β-thalassemia, is primarily caused by defects in the β-globin gene. The primary cause of β-thalassemia is an imbalance in the ratio of globin chains, with excess unbound α-globin chains deposited on red blood cell membranes, inhibiting the maturation of erythrocyte precursors, ultimately leading to ineffective erythrocyte production and a deficiency anemia. In the bone marrow of patients with β-thalassemia major, the proliferation of erythroid precursor cells leads to expansion of the bone marrow cavity and even deformities of the skull, facial bones, and long bones. Consequently, patients with β-thalassemia major have a distinctive facial appearance characterized by an enlarged head, prominent forehead, slightly high zygomatic arches, a low nasal bridge, widened eyes, and puffy eyelids. Patients also experience mottling of the skin, loss of appetite, growth retardation, and progressive hepatosplenomegaly. Among patients with compound heterozygous β-thalassemia, those with hereditary fetal hemoglobinosis (HPFH) often present with milder clinical manifestations. Studies have demonstrated that high levels of fetal hemoglobin (HbF) and α2γ2 in these patients compensate for the deficiencies of adult hemoglobin (HbA) and α2β2 in thalassemia patients, as synthesized γ-globin chains bind to excess α-globin chains. Therefore, reactivating fetal γ-globin expression and reducing free α-globin are crucial for the treatment of β-thalassemia.

[0003] B-cell chronic lymphocytic leukemia 11A (BCL11A) plays a crucial role in suppressing the γ-globin gene. A genome-wide association study (GWAS) revealed a strong association between BCL11A and abnormal fetal hemoglobin expression in patients with β-thalassemia. BCL11A, located on chromosome 2, is a zinc finger transcription factor that primarily suppresses γ-globin expression. When lentiviral-mediated shRNA knocks down BCL11A expression by 90%, HbF expression in erythrocytes reaches 70% of total hemoglobin, effectively alleviating hemolytic anemia in Berkeley sickle cell mice. Gene editing of the BCL11A erythroid enhancer specifically knocks down BCL11A expression in erythrocytes, reactivating γ-globin expression and effectively correcting the erythrocyte abnormalities seen in thalassemia patients. At present, another common approach to activate γ-globin is to use gene editing to simulate HPFH mutations, such as gene editing to simulate the 7.2kb deletion of the δβ-Corfu region and the 13bp deletion on the proximal promoter of γ-globin, thereby inducing the expression of γ-globin. In recent years, with the development of gene editing technology, single-base mutation technology has shown unique advantages. The reference "Zeng J, Wu Y, Ren C, et al. Therapeutic base editing of human hematopoietic stem cells. Nat Med, 2020, 26 (4): 535-541. doi: 10.1038 / s41591-020-0790-y" mentioned: using hA3A (N57Q)-BE3 to correct the A>G mutation at position -28 of the HBB promoter, and then combining to destroy the erythroid enhancer of BCL11A, to achieve effective multiple editing and produce effective HbF expression in animal transplantation experiments. The above data all indicate that the activation of γ-globin shows great potential in effectively correcting β-thalassemia red blood cell abnormalities.

[0004] Currently, β-thalassemia is mainly treated by inducing activation of γ-globin expression with chemical drugs, such as the demethylating agent 5-azacytidine and histone acetylase inhibitors, including butyrate and hydroxyurea. 5-Azacytidine can be used according to the reference “Ley TJ, DeSimone J, Anagnou NP, et al. 5-azacytidine selectively increases gammaglobin synthesis in a patient with beta+thalassemia. N Engl J Med, 1982, 307(24): 1469-1475. doi: 10.1056 / NEJM198212093072401.”; butyrate can be used according to the reference “Musallam KM, Taher AT, Cappellini MD, et al. Clinical experience with fetal hemoglobin induction therapy in patients with b-thalassemia. Blood, 2013, 121(12): 2199-2212; quiz 2372. doi: 10.1182 / blood-2012-10-408021.”; hydroxyurea can be used according to the reference “Zeng YT, Huang SZ, Ren ZR, et al. Hydroxyurea therapy in beta-thalassaemia intermedia: improvement in hematological parameters due to enhanced beta-globin synthesis. Br J Haematol, 1995, 90(3): 557-563. doi: 10.1111 / j.1365-2141.1995.tb05584.x.” and “Zamani F, Shakeri R, Eslami SM, et al. Hydroxyurea therapy in 49 patients with major beta-thalassemia. Arch Iran Med, 2009, 12(3): 295-297.” Although these chemical reagents have significant effects in inducing γ-globin expression, they are not highly targeted, and long-term use can cause many side effects on the body, such as bone marrow suppression, damage to the erythroid differentiation potential of hematopoietic stem cells, and liver and kidney damage.

[0005] Therefore, there is an urgent need to provide a new strategy for the treatment of thalassemia. Summary of the Invention

[0006] In order to solve the problems of bone marrow suppression, damage to the erythroid differentiation potential of hematopoietic stem cells and liver and kidney damage in the treatment of β-thalassemia with chemical drugs, the present invention provides an application of RBM3 in the preparation of a preparation for treating thalassemia.

[0007] The technical solution adopted in the present invention is:

[0008] In a first aspect, the present invention provides an application of RBM3 in the preparation of a preparation for treating thalassemia, wherein the nucleotide sequence of the RBM3 is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5.

[0009] Preferably, the thalassemia is β-thalassemia.

[0010] A second aspect of the present invention provides a recombinant lentiviral vector for down-regulating the expression of RBM3.

[0011] Preferably, the recombinant lentiviral vector uses the pLKO.1 vector as a backbone vector and is inserted with any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.3.

[0012] The third aspect of the present invention provides a lentiviral venom prepared by the recombinant lentiviral vector.

[0013] A fourth aspect of the present invention provides a method for preparing the lentiviral venom, comprising the following steps:

[0014] The recombinant lentiviral vector is transfected into cells, cultured, and the supernatant is collected to obtain lentiviral venom.

[0015] In a fifth aspect, the present invention provides a medicine comprising the recombinant lentiviral vector or the lentiviral venom.

[0016] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0017] Preferably, the pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

[0018] Preferably, the diluent includes any one of starch, lactose, sucrose and mannitol.

[0019] Preferably, the disintegrant includes any one of starch, microcrystalline cellulose and low-substituted hydroxypropyl cellulose.

[0020] Preferably, the precipitation inhibitor comprises any one of sodium lauryl sulfate, Tween-80, polyvinyl pyrrolidone and hydroxypropyl methylcellulose.

[0021] Preferably, the glidant includes any one of cationic polyacrylamide, polydiallyldimethylammonium chloride and cationic starch.

[0022] Preferably, the binder comprises any one of starch slurry, hydroxypropyl methylcellulose and povidone.

[0023] Preferably, the dispersant includes any one of sodium lauryl sulfate, polyvinyl pyrrolidone and sodium carboxymethyl cellulose.

[0024] Preferably, the suspending agent comprises any one of gum arabic, gum tragacanth, sodium carboxymethylcellulose and hydroxypropyl methylcellulose.

[0025] Preferably, the isotonic agent includes any one of sodium chloride, glucose and mannitol.

[0026] Preferably, the thickener includes any one of gum arabic, xanthan gum and sodium carboxymethyl cellulose.

[0027] Preferably, the emulsifier includes any one of sodium lauryl sulfate, benzalkonium chloride and sorbitan fatty acid.

[0028] Preferably, the preservative includes any one of benzoic acid, sorbic acid, methyl paraben and benzalkonium bromide.

[0029] Preferably, the stabilizer comprises any one of sodium sulfite, sodium bisulfite, tocopherol and disodium edetate.

[0030] Preferably, the pharmaceutically acceptable dosage form includes one of tablets, capsules, granules, injections, pills and powders.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a use of RBM3 in the preparation of a preparation for treating thalassemia. The nucleotide sequence of RBM3 is shown in SEQ ID NO. 4, and the amino acid sequence is shown in SEQ ID NO. 5. The present invention first discovered that downregulating RBM3 gene expression can significantly activate γ-globin expression, and that RBM3-related shRNA has a positive effect in the preparation of a treatment for β-thalassemia. Furthermore, during the experimental process of the present invention, no adverse reactions of RBM3 such as bone marrow suppression, impairment of the erythroid differentiation potential of hematopoietic stem cells, or liver and kidney damage were found. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 RBM3 silencing promotes γ-globin reactivation in HUDEP-2 cells. A: qPCR analysis of RBM3 mRNA levels. B: Western blot analysis of RBM3 protein levels. C: qPCR analysis of γ-globin expression levels. D: qPCR analysis of β-globin expression levels. E: Western blot analysis of γ-globin and β-globin protein levels.

[0034] Figure 2 Overexpression of RBM3 significantly inhibits γ-globin expression in HUDEP-2 cells. A: qPCR analysis of RBM3 mRNA expression. B: Western blot analysis of RBM3 protein expression. C: qPCR analysis of γ-globin mRNA expression. D: qPCR analysis of β-globin mRNA expression. E: Western blot analysis of γ-globin and β-globin protein expression.

[0035] Figure 3 RBM3 significantly regulates the expression of BCL11A, a transcriptional repressor of fetal hemoglobin. A: When RBM3 is downregulated, qPCR detected a significant downregulation of BCL11A mRNA levels. B: When RBM3 is downregulated, Western blot detected a significant downregulation of BCL11A protein levels. C: When RBM3 is upregulated, Western blot detected an upregulation of BCL11A protein levels in response to RBM3 upregulation. DETAILED DESCRIPTION

[0036] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0037] The inventive concept of the present invention is as follows:

[0038] The primary cause of β-thalassemia is an imbalance in the ratio of globin chains, with excess unbound α-globin chains deposited on the red blood cell membrane, inhibiting the maturation of erythrocyte precursors and ultimately leading to ineffective erythropoiesis and ischemic anemia. Currently, β-thalassemia is primarily treated with chemotherapy drugs that induce γ-globin expression, such as the demethylating agent 5-azacytidine and histone acetylase inhibitors, including butyrate and hydroxyurea. While these agents are highly effective in inducing γ-globin expression, they are not highly targeted, and long-term use can cause numerous side effects, including bone marrow suppression, impairment of the erythroid differentiation potential of hematopoietic stem cells, and liver and kidney damage.

[0039] Based on this, the present invention provides an application of RBM3 in the preparation of a preparation for treating thalassemia, wherein the nucleotide sequence of the RBM3 is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5.

[0040] RBM3 is an important RNA-binding protein, known as a survival gene for its resistance to serum deprivation, endoplasmic reticulum stress, and other adverse conditions. RBM3 is reported to be closely associated with synaptic regeneration and structural remodeling, holding important implications for the treatment of neurodegenerative diseases. Recent studies have demonstrated a crucial role for RBM3 in neuroprotection and have demonstrated its ability to effectively protect against apoptosis induced by neurotoxins such as MPP+, rotenone, nitric oxide (NO), and ultraviolet light, providing theoretical support for RBM3 as a molecular target for neurodegenerative diseases. Furthermore, RBM3 plays a key role in regulating mRNA stability and posttranscriptional translation. For example, RBM3 can promote skeletal muscle thickening by increasing mRNA stability and translation. In recent years, research on RBM3 has primarily focused on neuroregeneration, neuroprotection, and tumor progression, while limited research has been conducted on the role of RBM3 in hemoglobin development and the regulation of fetal hemoglobin.

[0041] In order to make those skilled in the art better understand the technical solution of the present invention and be able to implement it, the present invention is further described below in conjunction with specific examples. In the description of the present invention, if not otherwise specified, the reagents used are all commercially available and the methods used are all conventional techniques in the art.

[0042] The abbreviations of the present invention are shown in Table 1.

[0043] Table 1 Abbreviations

[0044] Abbreviation Full name β-globin β-globin γ-globin γ-globin BCL11A B-cell CLL / LYMPH 11A

[0045] Example 1

[0046] The application of RBM3 in the preparation of a preparation for treating thalassemia is as follows:

[0047] (1) Downregulation of RBM3 expression significantly promoted the reactivation of fetal hemoglobin in HUDEP-2 cells.

[0048] To investigate the effect of RBM3 on fetal hemoglobin expression, three shRNAs targeting RBM3 were designed using website prediction. The sequences of these three shRNAs are shown in SEQ ID NOs. 1 to 3, designated shRBM3-1#, shRBM3-2#, and shRBM3-3#, respectively. Recombinant lentiviral vectors were constructed using shRBM3-1, shRBM3-2, and shRBM3-3, respectively, using the pLKO.1 vector as a backbone vector. These recombinant lentiviral vectors were then transfected into cells, cultured, and the supernatant collected to obtain lentiviral venom.

[0049] After HUDEP-2 cells were infected with the recombinant lentiviral vector and stably expressed three shRNAs, the results were shown in Figure 1 qPCR and Western blot experiments confirmed that RBM3 was significantly downregulated at both the mRNA and protein levels. Subsequently, qPCR and Western blot analysis revealed that fetal γ-globin expression was significantly activated, particularly at the protein level. Furthermore, adult β-globin was found to have a significant decrease in mRNA levels, but protein levels were largely unaffected, with β-globin (1#) and β-globin (3#) remaining largely unaffected, with β-globin (2#) showing a significant decrease. These results suggest that reduced RBM3 expression contributes to the reactivation of fetal hemoglobin in HUDEP-2 cells.

[0050] shRBM3-1, SEQ ID NO.1: 5'-CCCAGTACCTATAAGAAAT-3';

[0051] shRBM3-2, SEQ ID NO.2: 5'-CCGCTACTCAGGAGGAAATTA-3';

[0052] shRBM3-3, SEQ ID NO.3: 5'-AGTGGCAGGTATTATGACAGT-3'.

[0053] The nucleotide sequence of RBM3 is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5.

[0054] SEQ ID NO.4:

[0055] ATGTCCTCTGAAGAAGGAAAGCTCTTCGTGGGAGGGCTCAACTTTAACACCGACGAGCAGGCACTGGAAGACCACTTCAGCAGTTTCGGACCTATCTCTGAGGTGGTCGTTGTCAAGGACCGGGAGACTCAGCGGTCCAGGGGTTTTGGTTTCATCACCTTCACCAACCCAGAGCATGCTTCAGTTGCCATGAGAGCCATGAACGGAGAGTCTCTGGATGGTCGTCAGATCCGTGTGGATCATGCAGGCAAGTCTGCTCGGGGAACCAGAGGAGGTGGCTTTGGGGCCCATGGGCGTGGTCGCAGCTACTCTAGAGGTGGTGGGGACCAGGGCTATGGGAGTGGCAGGTATTATGACAGTCGACCTGGAGGGTATGGATATGGATATGGACGTTCCAGAGACTATAATGGCAGAAACCAGGGTGGTTATGACCGCTACTCAGGAGGAAATTACAGAGACAATTATGACAACTGA。

[0056] SEQ ID NO.5:

[0057] MSSEEGKLFVGGLNFNTDEQALEDHFSSFGPISEVVVVKDRETQRSRGFGFITFTNPEHASVAMRAMNGESLDGRQIRVDHAGKSARGTRGGGFGAHGRGRSYSRGGGDQGYGSGRYYDSRPGGYGYGYGRSRDYNGRNQGGYDRYSGGNYRDNYDN。

[0058] Figure 1RBM3 silencing promotes γ-globin reactivation in HUDEP-2 cells. A: 96 hours after shRNA virus infection, total cell RNA was extracted, and RBM3 mRNA levels were analyzed by qPCR. B: 96 hours after shRNA virus infection, total cell protein was extracted, and RBM3 protein levels were analyzed by Western blot. C and D: After stable low-expression of RBM3 in HUDEP-2 cells, the expression levels of γ-globin and β-globin were analyzed by qPCR. E: After stable low-expression of RBM3 in HUDEP-2 cells, the protein levels of γ-globin and β-globin were analyzed by Western blot. All data were analyzed using the mean ± SD method, and each analysis was repeated at least three times. **p < 0.01, ***p < 0.001, compared with the shNC group, which serves as the shRNA blank control group.

[0059] (2) Overexpression of RBM3 significantly inhibited the reactivation of fetal hemoglobin in HUDEP-2 cells.

[0060] To further verify the effect of RBM3 on fetal hemoglobin expression, the present invention used a lentiviral expression system to stably overexpress RBM3 in HUDEP-2 cells. Figure 2 qPCR and Western blot experiments confirmed that RBM3 was significantly overexpressed in HUDEP-2 cells. Subsequent testing revealed that overexpression of RBM3 significantly inhibited γ-globin expression, while β-globin expression remained virtually unchanged. These results suggest that overexpression of RBM3 significantly inhibits the reactivation of fetal hemoglobin in HUDEP-2 cells.

[0061] Figure 2Overexpression of RBM3 significantly inhibits γ-globin expression in HUDEP-2 cells. A: 96 hours after HUDEP-2 cells were infected with RBM3 lentivirus, total RNA was extracted, and RBM3 mRNA expression was assessed by qPCR. B: 96 hours after HUDEP-2 cells were infected with RBM3 lentivirus, total protein was extracted, and RBM3 protein expression was assessed by Western blot. C and D: After stable overexpression of RBM3 in HUDEP-2 cells, γ-globin and β-globin mRNA expression levels were assessed by qPCR. E: After stable overexpression of RBM3 in HUDEP-2 cells, γ-globin and β-globin protein expression levels were assessed by Western blot. All data are analyzed as mean ± SD, and each analysis was performed in triplicate. **p < 0.01, ***p < 0.001 compared with the Vec group (Vec represents the virus-infected group).

[0062] (3) RBM3 regulates fetal hemoglobin expression by targeting BCL11A

[0063] To explore the mechanism by which reducing RBM3 expression can reactivate fetal hemoglobin expression, the present invention detected the star transcription factor BCL11A that inhibits fetal hemoglobin expression. Figure 3 , found that BCL11A was significantly downregulated at both mRNA and protein levels following RBM3 downregulation. Furthermore, RBM3 overexpression experiments confirmed that BCL11A was upregulated following RBM3 upregulation. Taken together, these results suggest that RBM3 may regulate γ-globin expression by modulating BCL11A expression.

[0064] Figure 3 RBM3 significantly regulates the expression of BCL11A, a transcriptional repressor of fetal hemoglobin. A: In HUDEP-2 cells with RBM3 downregulation, qPCR detected a significant downregulation of BCL11A at the mRNA level. B: In HUDEP-2 cells with RBM3 downregulation, Western blot detected a significant downregulation of BCL11A at the protein level. C: In HUDEP-2 cells with RBM3 upregulation, Western blot detected an upregulation of BCL11A at the protein level in response to RBM3 upregulation. All data were analyzed using the mean ± standard deviation method, with at least three replicates for each analysis. **p < 0.01, ***p < 0.001 compared with the shNC group.

[0065] The qPCR primer sequences of the present invention are shown in Table 2.

[0066] Table 2 qPCR primer sequence list

[0067]

[0068]

[0069] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0070] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. The use of RBM3 in the preparation of a preparation for treating thalassemia, characterized in that: The nucleotide sequence of RBM3 is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.

5.

2. The use according to claim 1, characterized in that The thalassemia is β-thalassemia.

3. A recombinant lentiviral vector for down-regulating the expression of RBM3 according to claim 1.

4. The recombinant lentiviral vector according to claim 3, characterized in that The recombinant lentiviral vector uses the pLKO.1 vector as a backbone vector and is inserted with any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.

3.

5. A lentiviral venom prepared using the recombinant lentiviral vector according to claim 3.

6. The method for preparing the lentiviral venom according to claim 5, characterized in that: The following steps are involved: The recombinant lentiviral vector is transfected into cells, cultured, and the supernatant is collected to obtain lentiviral venom.

7. A drug comprising the recombinant lentiviral vector according to claim 3 or the lentiviral venom according to claim 5.

8. The drug according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that The pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

10. The drug according to claim 7, characterized in that The pharmaceutically acceptable dosage form includes one of tablets, capsules, granules, injections, pills and powders.

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