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Androgen receptor antisense oligonucleotides

A peptide nucleic acid and polymer technology, applied in the field of peptide nucleic acid derivatives, can solve the problem of no androgenetic alopecia local treatment, and achieve the effect of good cell permeability and reduced risk of cross-reactivity

Active Publication Date: 2019-07-30
OLIPASS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, AR ASOs have been used in few topical treatments for androgenetic alopecia

Method used

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  • Androgen receptor antisense oligonucleotides
  • Androgen receptor antisense oligonucleotides
  • Androgen receptor antisense oligonucleotides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0328] Example 1. Exon skipping induced by "ASO 5"

[0329] "ASO 5" designated in Table 1 is a 13-mer antisense oligonucleotide that binds to a 20-mer RNA sequence [(5'→3')GC CUUGCCUG ┃ guaag gaaaa] The 13-mer sequence marked "bold" and "underlined" has a 13-mer full complementary overlap, and the 20-mer RNA sequence spans "exon 5" within the human AR pre-mRNA and the junction of "intron 5".

[0330] The ability of "ASO 5" to induce AR "exon 5" skipping in MCF7 cells (Cat. Number: HTB-22, ATCC) was evaluated by AR nested PCR. The procedure employed is detailed below.

[0331] [Cell culture and ASO treatment] at 37°C, in 5% CO 2 MCF7 cells were grown in EMEM medium supplemented with 10% FBS, 1% streptomycin / penicillin and 0.01 mg / ml bovine insulin under atmosphere. Cells were subcultured in 60 mm dishes and then treated with "ASO 5" at 3 aM-3 fM.

[0332] [RNA extraction] MCF7 cells were incubated with or without "ASO 5" for 3 hours. Total RNA was extracted from cells ...

Embodiment 2

[0336] Example 2. qPCR assessment of AR mRNA levels in MCF7 cells treated with "ASO 5"

[0337] The ability of "ASO 5" to downregulate human AR mRNA was assessed by qPCR with SYBR green detection.

[0338] MCF7 cells were subcultured in 5 mL medium in 60 mm dishes and treated with "ASO 5" at 0 zM (negative control)-1aM (2 dishes / each concentration). After 5 hours, total RNA was extracted with "MiniBEST Universal RNA Extraction Kit" according to the manufacturer's instruction (Cat. No. 9767, Takara). Following the manufacturer's instructions (Cat. No.6110A, Takara), 500 ng of RNA template was used to synthesize cDNA for a 50 μL reverse transcription reaction (using Oligo-dT). Then for a set of primers covering "exon 3" to "exon 9" [exon 3_forward: (5' → 3')TGGGTGTCACTATGGAGC, and exon 9_reverse: (5' → 3') GGGTG-TGGAAATAGAT-GGG], the cDNA was subjected to the first PCR, and the cycle conditions were as follows: 94°C for 2 min, followed by 15 cycles of 94°C for 15 sec, 55°C for...

Embodiment 3

[0341] Example 3. qPCR assessment of AR mRNA levels in MCF cells treated with "ASO 1"

[0342] Although "ASO 1" designated in Table 1 is a 13-mer antisense oligonucleotide, it was originally designed to complementarily target the junction of "exon 5" and "exon 6" within human AR mRNA. "ASO 1" with a 20-mer RNA sequence at the junction of "exon 5" and "intron 5" within human AR pre-mRNA [(5'→3')GC CUUGCCUG ┃ g The 9-mer sequences marked "bold" and "underlined" in "uaag" gaaaa] have 9-mer complementary overlaps. Notably, 4 single mismatches in intron 5 were marked as "uaag". Therefore, "ASO 1" can be regarded as an antisense oligonucleotide targeting human AR pre-mRNA, although only the 9-mer complementary overlaps in the 13-mer sequence.

[0343] The ability of "ASO 1" to downregulate human AR mRNA was assessed by qPCR according to the protocol described in "Example 2".

[0344] Figure 9 (B) provides the qPCR data obtained therefrom. When the ASO concentration was incre...

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Abstract

Provided are peptide nucleic acid derivatives targeting the 5' splice site of 'exon 5' within the human androgen receptor pre-mRNA. The peptide nucleic acid derivatives potently induce splice variantsof the androgen receptor mRNA in cells, and are useful to safely treat dermatological indications or conditions involving androgenic activity upon topical administration.

Description

[0001] The present invention relates to peptide nucleic acid derivatives targeting androgen receptor pre-mRNA for use in the treatment of dermatological indications or conditions mediated by androgen activity and claims U.S. Provisional Application No. 62 / 372035, which is hereby incorporated by reference in its entirety. [0002] Background of the invention [0003] Alopecia is a condition characterized by hair loss and thinning hair initially on the scalp. Androgenetic alopecia, also known as "male pattern baldness," is caused by pronounced androgenic activity in hair follicles and surrounding tissues. [0004] Although androgenetic alopecia affects both men and women, the condition tends to manifest differently in men and women. Men may experience alopecia areata, while women are more likely to experience generalized hair thinning on the scalp. The prevalence of androgenetic alopecia in men aged 30-50 is approximately 58% [ J. Invest. Dermatol . vol 9, 296-300(1997)]. An...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00A61K38/00
CPCA61K38/00C07K14/00C07K14/003C12N15/1138C12N2310/11C12N2310/3181C12N2310/33C12N2310/3513C12N2320/33C12N15/111A61P17/14A61P43/00Y02P20/55A61K9/0014A61K38/10A61K38/16C07K7/02C12N2320/32
Inventor 郑信郑多览曹峰准张降愿尹兴植
Owner OLIPASS CORP
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