Set of antisense oligonucleotides for specifically inhibiting 5α-reductase and application thereof

Abstract

Disclosed is a set of antisense oligonucleotides for specifically inhibiting 5α-reductase and an application thereof, wherein the antisense oligonucleotides have a length of 16-20 bases, and the antisense oligonucleotides (ASO) have the following applications: (1) preparation of a formulation for inhibiting 5α-reductase expression levels; and (2) preparation of a drug or a pharmaceutical composition for treating androgen-induced hair loss.

Description

A group of antisense oligonucleotides that specifically inhibit 5α-reductase and their applications Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a group of antisense oligonucleotides that specifically inhibit 5α-reductase and their applications. Background Technology

[0002] The epidemiological population for hair loss is increasing, with androgenetic alopecia being the most prevalent type. The incidence rate is 50% for men at age 50, with a lifetime incidence rate of 90%, while the lifetime incidence rate for women is 50%. However, treatment options are limited, with poor efficacy and significant side effects. Novel nucleic acid drugs represent a potential treatment for this type of skin condition. However, due to the current lack of effective technologies for delivering nucleic acid drugs into the skin, developing novel transdermal absorption nucleic acid drug (and even protein and small molecule drugs) delivery systems will expand the methods for treating skin diseases and is of great significance for drug development in the treatment of androgenetic alopecia.

[0003] 5α-reductase can convert testosterone and other types of androgens into the more potent androgen dihydrotestosterone (DHT). The newly generated DHT has a significantly higher affinity for the androgen receptor (AR) than testosterone, effectively activating the AR and inducing various skin diseases. Multiple experiments have confirmed that the androgen-androgen receptor axel is the most important regulatory mechanism for hair growth and a key pathway in the treatment of androgenetic alopecia (AGA). 5α-reductase and the androgen receptor are the two most crucial proteins in this signaling axis. There are three isoenzymes of 5α-reductase (5α-RI, RII, and RIII). RI and RII are the most comprehensively studied. RI is expressed in various cells of skin tissue and is universally expressed, while RII is expressed only in parts of the hair follicle, such as the inner root sheath, and at a higher level than RI. For androgenetic alopecia, histochemical analysis and gene knockout experiments have confirmed that RII is the more critical isoenzyme in the treatment of hair loss. Clinical trials have shown that drugs that act on both RI and RII can more effectively reduce DHT levels and have better overall efficacy.

[0004] Currently, targeted therapy for androgenetic alopecia mainly uses small molecule inhibitors, but the efficacy is limited and there are obvious side effects. Nucleic acid drugs, on the other hand, are a new type of treatment with the advantages of high specificity, significant efficacy, and low toxicity. They have great development potential and are expected to become an effective means of treating androgenetic alopecia.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] This invention first relates to a set of antisense oligonucleotides (ASOs) that specifically inhibit type I and type II 5α-reductases. The antisense oligonucleotides are 16-20 bases in length, and their target genes are SRD5A1 mRNA or pre-mRNA and SRD5A2 mRNA. The ASOs specifically pair with a specific region of the target gene, and the initiation site of the specific region is located at:

[0007] SRD5A1 pre-mRNA

[0008] (1)EXON1_INTRON1-2:570-575;

[0009] (2)EXON3_INTRON3-4:22880-22881;

[0010] (3)INTRON1-2: 781-782, 1155-1563, 3205, 3723-3725, 3845, 4184-4185, 7520-7716, 8001 -8005, 8724, 9856-9858, 10475-10485, 10781, 10999, 11909-11916, 13205, 15633-15638;

[0011] (4)INTRON1-2 INTRON2-3: 21021-21024, 7331-7334;

[0012] (5)INTRON2-3: 18977, 20069, 20489-20504, 20550, 21146-21148, 22084, 22115;

[0013] (6)INTRON2-3 EXON5 3UTR: 39221-39225, 21379-21383;

[0014] (7)INTRON2-3 INTRON3-4: 25543 19304, 25015 19564;

[0015] (8)INTRON3-4: 22976-23402, 24493, 25099-25110, 26299-26315, 26657-26658, 26802, 27162, 27351, 27916, 28213-28224

[0016] (9)INTRON3-4 INTRON4-5:32925-32928 25891-25894

[0017] (10)INTRON4-5:31477-31482, 31805, 33509, 34040-34075, 34431

[0018] (11)EXON5 3UTR: 37917, 37918, 38736, 38986~38988, 40395, 40401

[0019] SRD5A1 mRNA

[0020] (1)EXON5 3UTR: 2203-2235, 2665-2672, 3301-3422, 3537-3863, 2820-2909, 4129-4895, 5274, 6913, 37917-40401;

[0021] SRD5A2 mRNA

[0022] (1) 5UTR EXON1:18;

[0023] (2) EXON1: 21-32, 85-126, 192-203, 256-265;

[0024] (3) EXON1_2: 305-309;

[0025] (4) EXON2: 332-342, 407.408, 460, 461;

[0026] (5) EXON2 EXON2_3: 464-467;

[0027] (6) EXON3: 521-539;

[0028] (7) EXON3_4EXON4:579-587;

[0029] (8)EXON4_5EXON5:735-738,757-788;

[0030] (9)EXON5 3UTR:877-907, 1024-1086, 1100-1697, 2074-2295, 2467-2699, 2891-4456

[0031] The target gene of the SRD5A1 mRNA is numbered ENST00000274192.7, and its sequence is shown in SEQ ID NO.553.

[0032] The target gene of the SRD5A1 Pre-mRNA is numbered ENSG00000145545, and its sequence is shown in SEQ ID NO.554.

[0033] The target gene of the SRD5A2 mRNA is numbered ENST00000622030.2, and its sequence is shown in SEQ ID NO.555.

[0034] Furthermore, the ASO contains chemical modifications;

[0035] Preferably, the modification is: thio- or methoxyethyl modification (MOE modification) of the phosphate ester bond and 5-methyl modification of cytosine;

[0036] More preferably, the modification is as follows: all phosphate ester bonds of the ASO sequence are monothiolated, and 3 to 5 bases at the 3' and 5' ends are symmetrically modified with MOE and 5-methylated on all cytosine.

[0037] Furthermore, the ASO of the target gene, which is the mRNA or pre-mRNA of SRD5A1, is selected from any of the oligonucleotide sequences shown in Table 1 below (a total of 329 ASOs).

[0038] Table 1 (The motifs in the table indicate the modification positions; for example, 5-10-5 indicates that there are 5 bases at both the 3' and 5' ends that have undergone MOE modification.)

[0039] The ASO for the target gene SRD5A2 mRNA is selected from any of the oligonucleotide sequences shown in Table 2 below (a total of 217 ASOs);

[0040] Table 2 (The motifs in the table indicate the modification positions; for example, 5-10-5 indicates that there are 5 bases at the 3' end and 5' end that have undergone MOE modification.)

[0041] Preferably, all or part of the nucleotide modifications in the ASO described in the table are: monothiolation of the phosphate ester bond, methoxyethyl modification at the 2' position of the base (2'MOE modification), and 5-methyl modification of cytosine;

[0042] More preferably, the modification includes: monothiolation of phosphate ester bonds of all nucleotides in the ASO sequence, 2'-MOE modification of 3 to 5 bases at the 3' and 5' ends respectively, and 5-methyl modification of all cytosine.

[0043] After modification, the base sequence of the ASO is 4-8-4, 4-9-4, 5-8-5, 5-10-5, 5-12-5.

[0044] Furthermore, the present invention also relates to the following applications of the said antisense oligonucleotide (ASO) or its chemically modified forms:

[0045] (1) Prepare a formulation that inhibits the expression of 5α reductase protein;

[0046] (2) To prepare a drug or drug composition for treating hair loss caused by 5α-reductase; or

[0047] (3) Treatment for hair loss caused by 5α-reductase; or

[0048] (3) Inhibit the expression of 5α reductase protein.

[0049] The drug or pharmaceutical composition comprises a therapeutically effective amount of the antisense oligonucleotide (ASO), and necessary pharmaceutical excipients or delivery carriers.

[0050] The present invention also relates to a pharmaceutical composition comprising the aforementioned antisense oligonucleotide (ASO) or its chemically modified form, the pharmaceutical composition comprising: a therapeutically effective amount of the aforementioned antisense oligonucleotide (ASO) or its modified form, and necessary pharmaceutical excipients or delivery carriers; preferably, the pharmaceutical composition is a transdermal drug delivery formulation. Detailed Implementation

[0051] Example 1: Design, synthesis and modification of ASO targeting 5α-reductase target genes

[0052] The SRD5A1 mRNA (ENST00000274192.7) from the Ensembl database was selected as the target gene. The mRNA sequence of the target gene is shown in SEQ ID NO.553.

[0053] SEQ ID NO.553 (mRNA):

[0054] The SRD5A1 Pre-mRNA (ENSG00000145545) from the Ensembl database was selected as the target gene, with the sequence shown in SEQ ID NO. 554. SEQ ID NO. 554 (Pre-mRNA, containing introns):

[0055] The SRD5A2 mRNA (ENST00000622030.2) from the Ensembl database was selected as the target gene, with the sequence shown in SEQ ID NO.555. SEQ ID NO.555 (mRNA):

[0056] For SRD5A1 mRNA and Pre-mRNA, 329 ASO sequences were designed and selected for synthesis and in vitro efficacy testing. The specific locations and information of these ASOs are shown in Table 3 below. For SRD5A2 mRNA, 217 ASO sequences were designed and selected for synthesis and in vitro efficacy testing. The specific locations and information of these ASOs are shown in Table 4 below.

[0057] Furthermore, chemical modifications were performed on 563 of the aforementioned ASO sequences.

[0058] The specific modification methods are: monothiolation of phospholipid bonds of all or part of the nucleotides in the sequence, and MOE modification of 3 to 5 bases at the 3' and 5' ends respectively (the motifs in the table indicate the modification positions, for example, 5-10-5 means that 5 bases at the 3' and 5' ends are each modified with MOE).

[0059] Table 3. Specific information about the SRD5A1 ASO molecule.

[0060] Table 4. Detailed information on the SRD5A2 ASO molecule.

[0061] Example 2: Testing ASO activity using an in vitro cell model (PC-3 human prostate cancer cells).

[0062] In this embodiment, the inhibitory effect of the ASO molecules listed in Table 1 on the expression of type I 5α reductase (SRD5A1) in PC-3 cells was verified. The specific experimental procedure is as follows:

[0063] (1) Preparation of suspension transfection reagent: Dissolve ASO dry powder in sterile water to a concentration of 10 μM. Dilute 10 μM ASO stock solution to the required concentration using serum-reduced medium (basalmedia, L530KJ). Dilute Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019) with serum-reduced medium. Mix the transfection reagent dilution and ASO dilution separately to prepare ASO transfection complexes of the preset concentration or concentration gradient. Mix by pipetting and aspiration 10 times and let stand at room temperature for 20 min.

[0064] (2) Cell treatment: Cells were plated one day before transfection at a concentration of 5 × 10⁶ cells / mL. 3 Cells were seeded into 96-well plates, and 100 μl of F-12K medium containing 10% FBS was added to each well. Before transfection, the confluence of PC-3 cells should be >70% under a microscope. The old medium was discarded and replaced with 50 μl of F-12K medium containing 10% FBS. The ASO transfection complex prepared in step (1) was added to the 96-well plates and incubated at 37°C in a 5% CO2 incubator. After 5 hours, the medium was changed to fresh medium.

[0065] (3) 24 h after transfection, total RNA was extracted from the cells and the expression of SRD5A1 mRNA in the cells was detected by real-time quantitative PCR (Q-RT PCR). The qPCR primers and probes used to amplify the internal reference gene Actin and the target gene SRD5A1 are shown in Table 5-1.

[0066] Table 5-1 qPCR primer and probe sequences

[0067] Relative gene expression was calculated using the 2^-ΔΔCT method (Livak method). The inhibition rate of antisense oligonucleotide mRNA expression was calculated using the following equation: Inhibition rate = (1 - 2^-ΔΔCT) × 100%.

[0068] The experimental groups are as follows:

[0069] Cells treated with modified ASO as indicated by their respective numbers;

[0070] Blank, the blank control group, consists of cells that have not undergone any ASO treatment.

[0071] The efficiency of the ASO sequences described in Table 1 in inhibiting SRD5A1 mRNA in PC-3 cells is shown in Table 5-2 below. The results show that the ASO sequences shown in Table 1 have a significant inhibitory effect on SRD5A1 mRNA transcription.

[0072] Table 5-2 Inhibition rate (%) of SRD5A1 mRNA in PC-3 cells after treatment with antisense oligonucleotides

[0073] Example 3: Testing ASO activity in an in vitro cell model (PC-3 human prostate cancer cells stably expressing SRD5A2).

[0074] In this embodiment, a PC-3 cell line stably expressing the SRD5A2 CDS region sequence was first constructed using a lentiviral method. The inhibitory effect of the ASO molecules listed in Table 2 on the expression of type II 5α reductase (SRD5A2) in PC-3 cells stably expressing the SRD5A2 gene was verified. The specific experimental procedure is as follows:

[0075] (1) Preparation of suspension transfection reagent: Dissolve ASO dry powder in sterile water to a concentration of 10 μM. Dilute 10 μM ASO stock solution to the required concentration using serum-reduced medium (basalmedia, L530KJ). Dilute Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019) with serum-reduced medium. Mix the transfection reagent dilution and ASO dilution separately to prepare ASO transfection complexes of the preset concentration or concentration gradient. Mix by pipetting and aspiration 10 times and let stand at room temperature for 20 min.

[0076] (2) Cell treatment: Cells were plated one day before transfection at a concentration of 5 × 10⁶ cells / mL. 3 Cells were seeded into 96-well plates, and 100 μl of F-12K medium containing 10% FBS and 4 μg / ml puromycin was added to each well. Before transfection, the confluence of PC-3 cells stably expressing SRD5A2 should be >70% under a microscope. The old medium was discarded and replaced with 50 μl of F-12K medium containing 10% FBS. The ASO transfection complex prepared in step (1) was added to the 96-well plates and incubated at 37°C in a 5% CO2 incubator. After 5 h, the medium was changed to fresh medium.

[0077] (3) 24 h after transfection, total RNA was extracted from the cells and the expression of SRD5A2 mRNA in the cells was detected by real-time quantitative PCR (RT-qPCR). The qPCR primers and probes used to amplify the internal reference gene Actin and the target gene SRD5A2 are shown in Table 6-1.

[0078] Table 6-1 qPCR primer and probe sequences

[0079] Relative gene expression was calculated using the 2^-ΔΔCT method (Livak method), and the inhibition rate of antisense oligonucleotide mRNA expression level was calculated according to the following equation:

[0080] Inhibition rate = (1 - 2^-ΔΔCT) × 100%.

[0081] The experimental groups are as follows:

[0082] Cells treated with modified ASO as indicated by their respective numbers;

[0083] Blank, the blank control group, consists of cells that have not undergone any ASO treatment.

[0084] The efficiency of the ASO sequences described in Table 2 in inhibiting SRD5A2 mRNA in SRD5A2 stably expressed PC-3 cells is shown in Table 6-2 below. The results show that the ASO sequences shown in Table 2 have a significant inhibitory effect on the transcription of SRD5A2 mRNA.

[0085] Table 6-2 Inhibition rate (%) of SRD5A2 mRNA in PC-3 cells stably expressing SRD5A2 after treatment with antisense oligonucleotides.

[0086] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

Claims

1. A group of antisense oligonucleotides (ASOs) that specifically inhibit type I and type II 5α-reductases, wherein the antisense oligonucleotides are 16-20 bases in length, and the target genes are SRD5A1 mRNA, pre-mRNA, and SRD5A2 mRNA. The ASOs specifically pair with a specific region of the target gene, and the start site of the specific region is located at: SRD5A1 pre-mRNA: (1)EXON1_INTRON1-2:570-575; (2)EXON3_INTRON3-4:22880-22881; (3)INTRON1-2: 781-782, 1155-1563, 3205, 3723-3725, 3845, 4184-4185, 7520-7716, 8001 -8005, 8724, 9856-9858, 10475-10485, 10781, 10999, 11909-11916, 13205, 15633-15638; (4)INTRON1-2 INTRON2-3: 21021-21024, 7331-7334; (5)INTRON2-3: 18977, 20069, 20489-20504, 20550, 21146-21148, 22084, 22115; (6)INTRON2-3 EXON5 3UTR: 39221-39225, 21379-21383; (7)INTRON2-3 INTRON3-4: 25543 19304, 25015 19564; (8)INTRON3-4: 22976-23402, 24493, 25099-25110, 26299-26315, 26657-26658, 26802, 27162, 27351, 27916, 28213-28224; (9)INTRON3-4 INTRON4-5: 32925-32928 25891-25894; (10)INTRON4-5:31477-31482, 31805, 33509, 34040-34075, 34431; (11)EXON5 3UTR: 37917, 37918, 38736, 38986~38988, 40395, 40401; SRD5A1 mRNA: (1)EXON5 3UTR: 2203-2235, 2665-2672, 3301-3422, 3537-3863, 2820-2909, 4129-4895, 5274, 6913, 37917-40401; SRD5A2 mRNA: (1) 5UTR EXON1:18; (2) EXON1: 21-32, 85-126, 192-203, 256-265; (3) EXON1_2: 305-309; (4) EXON2: 332-342, 407.408, 460, 461; (5) EXON2 EXON2_3: 464-467; (6) EXON3: 521-539; (7) EXON3_4EXON4:579-587; (8)EXON4_5EXON5:735-738,757-788; (9)EXON5 3UTR:877-907, 1024-1086, 1100-1697, 2074-2295, 2467-2699, 2891-4456; The sequence of the SRD5A1 mRNA is shown in SEQ ID NO.553; The sequence of the SRD5A1 Pre-mRNA is shown in SEQ ID NO.554; The sequence of the SRD5A2 mRNA is shown in SEQ ID NO.

555.

2. The antisense oligonucleotide according to claim 1, characterized in that, The ASO mentioned above is a chemically modified version containing chemical modifications; Preferably, the modification is: thio- or methoxyethyl modification (MOE modification) of the phosphate ester bond and 5-methyl modification of cytosine; More preferably, the modification is as follows: all phosphate ester bonds of the ASO sequence are monothiolated, and 3 to 5 bases at the 3' and 5' ends are symmetrically modified with MOE and 5-methylated on all cytosine.

3. The antisense oligonucleotide according to claim 1 or 2, characterized in that, The target gene is SRD5A1 mRNA and the ASO of pre-mRNA selected from any of the antisense oligonucleotides shown in Table 1 below; Table 1 Preferably, all or part of the nucleotide modifications in the ASO described in the table are: monothiolation of the phosphate ester bond, methoxyethyl modification at the 2' position of the base (2'MOE modification), and 5-methyl modification of cytosine; More preferably, the modification includes: monothiolation of phosphate ester bonds of all nucleotides in the ASO sequence, 2'-MOE modification of 3 to 5 bases at the 3' and 5' ends respectively, and 5-methyl modification of all cytosine. After modification, the base sequence of the ASO is 4-8-4, 4-9-4, 5-8-5, 5-10-5, 5-12-5.

4. The antisense oligonucleotide according to claim 1 or 2, characterized in that, The ASO of the target gene SRD5A2 mRNA is selected from any of the antisense oligonucleotides shown in Table 2 below; Table 2 Preferably, all or part of the nucleotide modifications in the ASO described in the table are: monothiolation of the phosphate ester bond, methoxyethyl modification at the 2' position of the base (2'MOE modification), and 5-methyl modification of cytosine; More preferably, the modification includes: monothiolation of phosphate ester bonds of all nucleotides in the ASO sequence, 2'-MOE modification of 3 to 5 bases at the 3' and 5' ends respectively, and 5-methyl modification of all cytosine. After modification, the base sequence of the ASO is 4-8-4, 4-9-4, 5-8-5, 5-10-5, 5-12-5.

5. The following applications of the antisense oligonucleotide (ASO) or its chemically modified form as described in any one of claims 1-4: (1) Prepare a formulation that inhibits the expression of 5α reductase protein; (2) To prepare a drug or drug composition for treating hair loss caused by 5α-reductase; or (3) Treatment for hair loss caused by 5α-reductase; or (4) Inhibit the expression of 5α reductase protein.

6. The application according to claim 5, characterized in that, The drug or pharmaceutical composition comprises a therapeutically effective amount of the antisense oligonucleotide (ASO) or its chemically modified form, and necessary pharmaceutical excipients or delivery carriers.

7. A pharmaceutical composition comprising any one of the antisense oligonucleotides (ASO) or their chemically modified forms according to claims 1-4, wherein the pharmaceutical composition comprises: The pharmaceutical composition comprises a therapeutically effective amount of the said antisense oligonucleotide (ASO) or its modified form, and necessary pharmaceutical excipients or delivery carriers; preferably, the pharmaceutical composition is a transdermal delivery formulation.

Images