JPWO2021211325A5 - - Google Patents
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- JPWO2021211325A5 JPWO2021211325A5 JP2022562629A JP2022562629A JPWO2021211325A5 JP WO2021211325 A5 JPWO2021211325 A5 JP WO2021211325A5 JP 2022562629 A JP2022562629 A JP 2022562629A JP 2022562629 A JP2022562629 A JP 2022562629A JP WO2021211325 A5 JPWO2021211325 A5 JP WO2021211325A5
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- Prior art keywords
- polynucleotide
- fusion polypeptide
- sgrna
- seq
- present
- Prior art date
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- 108091033319 polynucleotide Proteins 0.000 claims description 23
- 102000040430 polynucleotide Human genes 0.000 claims description 23
- 239000002157 polynucleotide Substances 0.000 claims description 23
- 230000004927 fusion Effects 0.000 claims description 19
- 229920001184 polypeptide Polymers 0.000 claims description 19
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 19
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 19
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 18
- 239000013598 vector Substances 0.000 claims description 15
- 108091033409 CRISPR Proteins 0.000 claims description 14
- 230000001973 epigenetic effect Effects 0.000 claims description 10
- 238000010446 CRISPR interference Methods 0.000 claims description 8
- 102100021158 Double homeobox protein 4 Human genes 0.000 claims description 6
- 208000037149 Facioscapulohumeral dystrophy Diseases 0.000 claims description 6
- 101000968549 Homo sapiens Double homeobox protein 4 Proteins 0.000 claims description 6
- 208000008570 facioscapulohumeral muscular dystrophy Diseases 0.000 claims description 6
- 102000014669 Chromo shadow domains Human genes 0.000 claims description 4
- 108050005011 Chromo shadow domains Proteins 0.000 claims description 4
- 101150054841 DUX4 gene Proteins 0.000 claims description 4
- 102100028998 Histone-lysine N-methyltransferase SUV39H1 Human genes 0.000 claims description 4
- 101000696705 Homo sapiens Histone-lysine N-methyltransferase SUV39H1 Proteins 0.000 claims description 4
- 102000006890 Methyl-CpG-Binding Protein 2 Human genes 0.000 claims description 4
- 108010072388 Methyl-CpG-Binding Protein 2 Proteins 0.000 claims description 4
- 210000004899 c-terminal region Anatomy 0.000 claims description 4
- 230000014509 gene expression Effects 0.000 claims description 4
- 239000003112 inhibitor Substances 0.000 claims description 4
- 230000037426 transcriptional repression Effects 0.000 claims description 4
- 150000007523 nucleic acids Chemical group 0.000 claims description 3
- 108020005004 Guide RNA Proteins 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 208000035475 disorder Diseases 0.000 claims description 2
- 210000002363 skeletal muscle cell Anatomy 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 4
- 238000000034 method Methods 0.000 description 13
- 241000124008 Mammalia Species 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 2
- 210000002027 skeletal muscle Anatomy 0.000 description 2
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
Description
特定の態様において、哺乳動物はヒトである。
[本発明1001]
一本鎖ガイドRNA（sgRNA）と融合ポリペプチドとを含むCRISPR干渉（CRISPRi）プラットフォームをコードするポリヌクレオチドであって、前記融合ポリペプチドが、エピジェネティック抑制因子に融合された触媒不活性型Cas9（dCas9またはiCas9）をさらに含む、前記ポリヌクレオチド。
[本発明1002]
前記sgRNAがU6プロモーターの制御下にある、本発明1001のポリヌクレオチド。
[本発明1003]
前記sgRNAがDUX4遺伝子座を標的とする、本発明1001のポリヌクレオチド。
[本発明1004]
前記融合ポリペプチドが骨格筋特異的調節カセットの制御下にある、本発明1001～1003のいずれかのポリヌクレオチド。
[本発明1005]
前記触媒不活性型Cas9がdSaCas9である、本発明1001～1004のいずれかのポリヌクレオチド。
[本発明1006]
前記エピジェネティック抑制因子が、HP1α、HP1γ、HP1αまたはHP1γのクロモシャドウドメインおよびC末端伸長領域、MeCP2転写抑制ドメイン（TRD）、ならびにSUV39H1 SETドメインからなる群より選択される、本発明1001～1005のいずれかのポリヌクレオチド。
[本発明1007]
前記sgRNAがSEQ ID NO:38、39、40、41、42、または43を含む、本発明1001～1006のいずれかのポリヌクレオチド。
[本発明1008]
前記融合ポリペプチドがSEQ ID NO:1～4のうちのいずれか1つを含む、本発明1001～1006のいずれかのポリヌクレオチド。
[本発明1009]
SEQ ID NO:48～55のうちのいずれか1つを含む、本発明1001～1006のいずれかのポリヌクレオチド。
[本発明1010]
sgRNAと融合ポリペプチドとを含むCRISPRiプラットフォームをコードするポリヌクレオチドを含むベクターであって、前記融合ポリペプチドが、エピジェネティック抑制因子に融合された触媒不活性型Cas9（dCas9またはiCas9）をさらに含む、前記ベクター。
[本発明1011]
前記sgRNAがU6プロモーターの制御下にある、本発明1010のベクター。
[本発明1012]
前記sgRNAがDUX4遺伝子座を標的とする、本発明1010のベクター。
[本発明1013]
前記融合ポリペプチドが骨格筋特異的調節カセットの制御下にある、本発明1010～1012のいずれかのベクター。
[本発明1014]
前記触媒不活性型Cas9がdSaCas9である、本発明1010～1013のいずれかのベクター。
[本発明1015]
前記エピジェネティック抑制因子が、HP1α、HP1γ、HP1αまたはHP1γのクロモシャドウドメインおよびC末端伸長領域、MeCP2転写抑制ドメイン（TRD）、ならびにSUV39H1 SETドメインからなる群より選択される、本発明1010～1014のいずれかのベクター。
[本発明1016]
前記sgRNAが、SEQ ID NO:38、39、40、41、42、または43を含む群より選択される核酸を含む、本発明1010～1015のいずれかのベクター。
[本発明1017]
前記融合ポリペプチドがSEQ ID NO:1～4のうちのいずれか1つを含む、本発明1010～1016のいずれかのベクター。
[本発明1018]
前記ポリヌクレオチドがSEQ ID NO:48～55のうちのいずれか1つを含む、本発明1010～1017のいずれかのベクター。
[本発明1019]
アデノ随伴ウイルス（AAV）ベクターである、本発明1010～1018のいずれかのベクター。
[本発明1020]
SEQ ID NO:48～55のうちのいずれか1つを含む、本発明1010～1019のいずれかのベクター。
[本発明1021]
その必要のある対象における顔面肩甲上腕型筋ジストロフィー（FSHD）を処置する方法であって、有効量の、DUX4遺伝子発現の抑制因子を、前記対象に投与する工程を含み、前記抑制因子が前記対象の骨格筋細胞におけるDUX4遺伝子発現を減少させ、それによって障害を処置する、前記方法。
[本発明1022]
前記DUX4抑制因子が、sgRNAと融合ポリペプチドとを含むCRISPRiプラットフォームを含むポリヌクレオチドであり、前記融合ポリペプチドが、エピジェネティック抑制因子に融合されたdCas9をさらに含む、本発明1021の方法。
[本発明1023]
前記sgRNAがDUX4遺伝子座を標的とする、本発明1021～1022のいずれかの方法。
[本発明1024]
前記sgRNAが、SEQ ID NO:38、39、40、41、42、または43からなる群より選択される核酸配列を含む、本発明1021～1023のいずれかの方法。
[本発明1025]
前記dCas9がdSaCas9である、本発明1021～1024のいずれかの方法。
[本発明1026]
前記エピジェネティック抑制因子が、HP1α、HP1γ、HP1αまたはHP1γのクロモシャドウドメインおよびC末端伸長領域、MeCP2転写抑制ドメイン（TRD）、ならびにSUV39H1 SETドメインからなる群より選択される、本発明1021～1025のいずれかの方法。
[本発明1027]
前記融合ポリペプチドが、SEQ ID NO:1～4のうちのいずれか1つを含むポリヌクレオチドによってコードされる、本発明1021～1026のいずれかの方法。
[本発明1028]
前記ポリヌクレオチドがSEQ ID NO:48～55のうちのいずれか1つを含む、本発明1021～1027のいずれかの方法。
[本発明1029]
前記対象が哺乳動物である、本発明1021～1028のいずれかの方法。
[本発明1030]
前記哺乳動物がヒトである、本発明1029の方法。
[本発明1031]
その必要のある対象におけるFSHDを処置する方法であって、有効量の、本発明1010～1020のいずれかのベクターを、前記対象に投与する工程を含む、前記方法。
[本発明1032]
前記対象が哺乳動物である、本発明1031の方法。
[本発明1033]
前記哺乳動物がヒトである、本発明1032の方法。
In certain embodiments, the mammal is a human.
[The present invention 1001]
1. A polynucleotide encoding a CRISPR interference (CRISPRi) platform comprising a single-stranded guide RNA (sgRNA) and a fusion polypeptide, the fusion polypeptide further comprising a catalytically inactive Cas9 (dCas9 or iCas9) fused to an epigenetic repressor.
[The present invention 1002]
The polynucleotide of the present invention, wherein the sgRNA is under the control of a U6 promoter.
[The present invention 1003]
The polynucleotide of any one of claims 1 to 10, wherein the sgRNA targets the DUX4 locus.
[The present invention 1004]
The polynucleotide of any of claims 1001 to 1003, wherein the fusion polypeptide is under the control of a skeletal muscle-specific regulatory cassette.
[The present invention 1005]
The polynucleotide of any one of claims 1001 to 1004, wherein the catalytically inactive Cas9 is dSaCas9.
[The present invention 1006]
The polynucleotide of any of claims 1001 to 1005, wherein the epigenetic repressor is selected from the group consisting of HP1α, HP1γ, the chromoshadow domain and C-terminal extension region of HP1α or HP1γ, the MeCP2 transcriptional repression domain (TRD), and the SUV39H1 SET domain.
[The present invention 1007]
The polynucleotide of any one of claims 1001 to 1006, wherein the sgRNA comprises SEQ ID NO:38, 39, 40, 41, 42, or 43.
[The present invention 1008]
The polynucleotide of any one of claims 1001 to 1006, wherein the fusion polypeptide comprises any one of SEQ ID NOs: 1 to 4.
[The present invention 1009]
Any of the polynucleotides of 1001 to 1006 comprising any one of SEQ ID NOs: 48 to 55.
[The present invention 1010]
A vector comprising a polynucleotide encoding a CRISPRi platform comprising an sgRNA and a fusion polypeptide, the fusion polypeptide further comprising a catalytically inactive Cas9 (dCas9 or iCas9) fused to an epigenetic repressor.
[The present invention 1011]
The vector of the present invention, wherein the sgRNA is under the control of a U6 promoter.
[The present invention 1012]
The vector of the present invention, wherein the sgRNA targets the DUX4 locus.
[The present invention 1013]
Any of the vectors of claims 1010 to 1012, wherein the fusion polypeptide is under the control of a skeletal muscle-specific regulatory cassette.
[The present invention 1014]
The vector of any one of claims 1010 to 1013, wherein the catalytically inactive Cas9 is dSaCas9.
[The present invention 1015]
The vector of any of claims 1010 to 1014, wherein the epigenetic repressor is selected from the group consisting of HP1α, HP1γ, the chromoshadow domain and C-terminal extension region of HP1α or HP1γ, the MeCP2 transcriptional repression domain (TRD), and the SUV39H1 SET domain.
[The present invention 1016]
The vector of any of claims 1010 to 1015, wherein the sgRNA comprises a nucleic acid selected from the group comprising SEQ ID NO:38, 39, 40, 41, 42, or 43.
[The present invention 1017]
The vector of any one of claims 1010 to 1016, wherein the fusion polypeptide comprises any one of SEQ ID NOs: 1 to 4.
[The present invention 1018]
The vector of any one of claims 1010 to 1017, wherein the polynucleotide comprises any one of SEQ ID NOs: 48 to 55.
[The present invention 1019]
Any of the vectors of 1010 to 1018, which is an adeno-associated virus (AAV) vector.
[The present invention 1020]
Any of the vectors of 1010 to 1019, comprising any one of SEQ ID NOs:48 to 55.
[The present invention 1021]
A method for treating facioscapulohumeral muscular dystrophy (FSHD) in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of DUX4 gene expression, wherein the inhibitor reduces DUX4 gene expression in skeletal muscle cells of the subject, thereby treating the disorder.
[The present invention 1022]
The method of claim 1021, wherein the DUX4 repressor is a polynucleotide comprising a CRISPRi platform comprising an sgRNA and a fusion polypeptide, and the fusion polypeptide further comprises dCas9 fused to an epigenetic repressor.
[The present invention 1023]
1023. The method of any of claims 1021 to 1022, wherein the sgRNA targets the DUX4 locus.
[The present invention 1024]
The method of any of claims 1021 to 1023, wherein the sgRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:38, 39, 40, 41, 42, or 43.
[The present invention 1025]
Any of the methods of claims 1021 to 1024, wherein the dCas9 is dSaCas9.
[The present invention 1026]
1026. The method of any of claims 1021 to 1025, wherein said epigenetic repressor is selected from the group consisting of HP1α, HP1γ, the chromoshadow domain and C-terminal extension region of HP1α or HP1γ, the MeCP2 transcriptional repression domain (TRD), and the SUV39H1 SET domain.
[The present invention 1027]
The method of any of claims 1021 to 1026, wherein said fusion polypeptide is encoded by a polynucleotide comprising any one of SEQ ID NOs: 1 to 4.
[The present invention 1028]
The method of any of claims 1021 to 1027, wherein the polynucleotide comprises any one of SEQ ID NOs: 48 to 55.
[The present invention 1029]
The method of any one of claims 1021 to 1028, wherein the subject is a mammal.
[The present invention 1030]
The method of claim 1029, wherein the mammal is a human.
[The present invention 1031]
A method for treating FSHD in a subject in need thereof, comprising administering to the subject an effective amount of any of the vectors of inventions 1010 to 1020.
[The present invention 1032]
The method of claim 1031, wherein the subject is a mammal.
[The present invention 1033]
The method of claim 1032, wherein the mammal is a human.
Claims (10)
前記融合ポリペプチドがSEQ ID NO:1～4のうちのいずれか1つを含む；および/または
前記ポリヌクレオチドがSEQ ID NO:48～55のうちのいずれか1つを含む、
請求項1～4のいずれか一項記載のポリヌクレオチド。 the sgRNA comprises SEQ ID NO:38, 39, 40, 41, 42, or 43 ;
the fusion polypeptide comprises any one of SEQ ID NOs:1-4; and/or
the polynucleotide comprises any one of SEQ ID NOs: 48-55;
A polynucleotide according to any one of claims 1 to 4 .
前記融合ポリペプチドが、SEQ ID NO:1～4のうちのいずれか1つを含むポリヌクレオチドによってコードされる、
請求項7または8記載の薬学的組成物。 the sgRNA comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, 41, 42, or 43; and/or
The fusion polypeptide is encoded by a polynucleotide comprising any one of SEQ ID NOs: 1 to 4;
9. The pharmaceutical composition of claim 7 or 8 .
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US202063011476P | 2020-04-17 | 2020-04-17 | |
| US63/011,476 | 2020-04-17 | ||
| PCT/US2021/025940 WO2021211325A1 (en) | 2020-04-17 | 2021-04-06 | Crispr-inhibition for facioscapulohumeral muscular dystrophy |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2023522020A JP2023522020A (en) | 2023-05-26 |
| JPWO2021211325A5 true JPWO2021211325A5 (en) | 2024-04-19 |
Family
ID=78084611
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2022562629A Pending JP2023522020A (en) | 2020-04-17 | 2021-04-06 | CRISPR inhibition for facioscapulohumeral muscular dystrophy |
Country Status (11)
| Country | Link |
|---|---|
| US (1) | US20230174958A1 (en) |
| EP (1) | EP4135778A4 (en) |
| JP (1) | JP2023522020A (en) |
| KR (1) | KR20230003511A (en) |
| CN (1) | CN115768487A (en) |
| AU (1) | AU2021257213A1 (en) |
| BR (1) | BR112022020945A2 (en) |
| CA (1) | CA3175625A1 (en) |
| IL (1) | IL297113A (en) |
| MX (1) | MX2022012965A (en) |
| WO (1) | WO2021211325A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20230023612A (en) | 2020-04-02 | 2023-02-17 | 마이레큘, 인크. | Targeted inhibition using engineered oligonucleotides |
| WO2024020444A2 (en) * | 2022-07-20 | 2024-01-25 | Nevada Research & Innovation Corporation | Muscle-specific regulatory cassettes |
| CN117448380A (en) * | 2023-12-22 | 2024-01-26 | 上海元戊医学技术有限公司 | Construction method and application of COL10A1 protein low-expression MSC cell strain derived from iPSC |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3636778B1 (en) * | 2010-08-18 | 2022-01-12 | Fred Hutchinson Cancer Research Center | Agents for use in treating facioscapulohumeral dystrophy (fshd) |
| CA2936726C (en) * | 2014-01-21 | 2024-04-30 | Vrije Universiteit Brussel | Muscle-specific nucleic acid regulatory elements and methods and use thereof |
| CA3074723A1 (en) * | 2016-09-23 | 2018-03-29 | University Of Massachusetts | Silencing of dux4 by recombinant gene editing complexes |
-
2021
- 2021-04-06 KR KR1020227039447A patent/KR20230003511A/en active Search and Examination
- 2021-04-06 AU AU2021257213A patent/AU2021257213A1/en active Pending
- 2021-04-06 BR BR112022020945A patent/BR112022020945A2/en unknown
- 2021-04-06 EP EP21787712.5A patent/EP4135778A4/en active Pending
- 2021-04-06 WO PCT/US2021/025940 patent/WO2021211325A1/en active Application Filing
- 2021-04-06 US US17/919,198 patent/US20230174958A1/en active Pending
- 2021-04-06 JP JP2022562629A patent/JP2023522020A/en active Pending
- 2021-04-06 MX MX2022012965A patent/MX2022012965A/en unknown
- 2021-04-06 CN CN202180041592.XA patent/CN115768487A/en active Pending
- 2021-04-06 CA CA3175625A patent/CA3175625A1/en active Pending
-
2022
- 2022-10-06 IL IL297113A patent/IL297113A/en unknown
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