JPWO2020183419A5

JPWO2020183419A5 -

Info

Publication number: JPWO2020183419A5
Application number: JP2021555368A
Authority: JP
Publication date: 2023-03-22

Claims

A method of producing an RNA molecule having silencing activity in a cell, comprising:
(a) identifying a nucleic acid sequence that encodes an RNA molecule, comprising:
i. the nucleic acid sequence exhibits a range of sequence homology, but not complete identity, to a nucleic acid sequence encoding an RNA molecule that associates with the RNA-induced silencing complex (RISC);
ii. the nucleic acid sequence is determined to be transcribable, and
iii. determining that the RNA molecule is aberrantly processed;
(b ) modifying said nucleic acid sequence to confer processability of said RNA molecule into a small RNA that associates with RISC and is complementary to a target RNA;
thereby producing an RNA molecule having silencing activity in the cell;
method including.

(i) the RNA molecule of step (a) has a defined range of sequence homology, but not complete identity, to an RNA molecule that associates with RISC and/or is processed into a molecule that associates with RISC; indicate and/or
(ii) conferring processability in step (b) is normal to the RNA molecule encoded by the nucleic acid sequence of said nucleic acid sequence encoding an RNA molecule that associates with the RNA-induced silencing complex (RISC); and/or
(iii) the method further comprises determining the genomic location of the nucleic acid sequence encoding the RNA molecule of step (a);
(I) the genomic location is within a non-coding gene, optionally within an intron of a non-coding gene, or
(II) said genomic location is within a coding gene, optionally within an exon of a coding gene, optionally within an exon encoding an untranslated region (UTR) of a coding gene, or optionally within an intron of a coding gene. be,
The method of claim 1.

(i) optionally, said small RNA, wherein step (a) is affected by alignment of expression data of said small RNA to the genome of said cell and determination of the amount of reads mapped to each genomic location; is to a predetermined position in the genome of the cell without mismatches; and/or
(ii) said modification in step (b) confers a structure to said abnormally processed RNA molecule such that said RNA molecule is processed into a small RNA that associates with RISC; and / or
(iii) said modification in step (b) is performed with a nucleic acid other than that corresponding to the binding site for said target RNA, and/or
(iv) said processivity is mediated by a cellular nuclease selected from the group consisting of Dicer, Argonaute, tRNA cleaving enzyme, and Piwi-associated RNA (piRNA)-related proteins;
3. A method according to claim 1 or 2 .

The modification in step ( b ) introduces into the cell a DNA editing agent that reactivates the silencing activity of said aberrantly processed RNA molecule towards said target RNA, thereby silencing within said cell. 4. The method of any one of claims 1-3 , comprising producing an RNA molecule having sing activity.

further comprising modifying the specificity of said RNA molecule that has silencing activity within a cell, wherein said DNA editing agent directs the silencing specificity of said RNA molecule towards a target RNA of interest that is different from said target RNA. , thereby modifying the specificity of the RNA molecule having the silencing activity in the cell.

The nucleic acid sequence encoding said RNA molecule identified in step (a) encodes a silencing RNA molecule whose silencing activity and/or processing into small silencing RNAs is dependent on its secondary structure. is homologous to the gene in
optionally,
(a) the silencing RNA molecule is microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA or U-RNA), small nucleolar RNA (snoRNA), small molecule Cajal body RNA (scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), repeat-derived RNA, autonomous and non-autonomous transposable and retrotransposable element-derived RNA, autonomous and non-autonomous transposition selected from the group consisting of transposable and retrotransposable element RNAs, and long noncoding RNAs (lncRNAs); or
(b) the modification of step (b) has a secondary structure substantially equivalent to that of the silencing RNA whose silencing activity and/or processing into small silencing RNA is dependent on that secondary structure; altering said sequence as said RNA molecule has;
A method according to any one of claims 1-5 .

A genetically modified cell comprising a genome comprising a polynucleotide sequence encoding an RNA molecule having a nucleic acid sequence change that causes the RNA molecule to be processed into a small RNA that associates with RISC, said cell comprising: A genetically modified cell wherein said processing of said RNA molecule is absent in a co-originating wild-type cell without said nucleic acid sequence alteration.

8. The genetically modified cell of Claim 7 , wherein the processing is standard processing.

said RNA molecule is microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), Piwi binding RNA (piRNA), phased small interfering RNA (phasiRNA), trans-acting siRNA (tasiRNA) ), transfer RNA fragment (tRF), small nuclear RNA (snRNA), transposable and/or retrotransposable RNA, autonomous and non-autonomous transposable and/or retrotransposable RNA. A method according to any one of claims 1 to 5 or a genetically modified cell according to claim 7 or 8, selected.

10. The method of any one of claims 1-6 or 9 , further comprising introducing a donor oligonucleotide into the cell.

(i) the DNA editing agent comprises at least one sgRNA;
optionally, said DNA editing agent comprises an endonuclease;
Optionally, said DNA editing agent is selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR endonucleases, dCRISPR endonucleases, and homing endonucleases. of the editing system,
optionally, said endonuclease comprises Cas9; or
(ii) the DNA editing agent is endonuclease-free, and/or
the DNA editing agent is applied to the cell as DNA, RNA, or RNP;
11. A method according to any one of claims 6, 9 or 10 .

said target RNA of interest is endogenous or exogenous to said cell, and/or
said specificity of said RNA molecule is cell size, growth rate/inhibition, cell shape, cell membrane integrity, tumor size, tumor shape, organism coloration, organism size, crop yield, metabolic profile, fruit traits, organism phenotypically determined by determining at least one phenotype selected from the group consisting of biotic stress resistance, abiotic stress resistance, infectious parameters, and inflammatory parameters;
A method according to any one of claims 5 or 9-11 .

said cells are eukaryotic cells;
optionally, said eukaryotic cell is obtained from a eukaryotic organism selected from the group consisting of plants, mammals, invertebrates, insects, nematodes, birds, reptiles, fish, crustaceans, fungi and algae;
optionally, said eukaryotic cells are plant cells, and
optionally, said plant cell is a protoplast
A method according to any one of claims 5 or 9-12 or a genetically modified cell according to any one of claims 7-9 .

A plant cell produced according to the method of any one of claims 1-6 or 9-13 .

A plant comprising the plant cell of claim 14 ,
Optionally, the plant, wherein said plant is non-transgenic .

A method of producing a plant with reduced expression of a target gene comprising:
(a) breeding a plant according to claim 15 ;
(b) selecting progeny plants with reduced expression of a target RNA of interest or progeny comprising silencing specificity in said RNA molecule for said target RNA of interest and free of said DNA editing agent;
thereby producing said plant with reduced expression of the target gene;
method including.

1. A method of producing a plant comprising an RNA molecule having silencing activity against a target RNA of interest, comprising:
(a) breeding a plant according to claim 15 ;
(b) selecting progeny plants comprising said RNA molecules having said silencing activity against said target RNA of interest or progeny comprising silencing specificity in said RNA molecules against said target RNA of interest and free of said DNA editing agent; death,
thereby producing a plant comprising an RNA molecule having silencing activity against a target RNA of interest;
method including.

16. A method of producing a plant or plant cell according to claim 15 , comprising growing the plant or plant cell under conditions permitting reproduction.

18. A method according to claim 16 or 17 , wherein said breeding comprises crossing or selfing.

Seed of a plant according to claim 15 or a plant produced according to any one of claims 16-19 .

said eukaryotic cells are human cells;
optionally, the nucleic acid sequence encoding the RNA molecule is selected from the group consisting of the nucleic acid sequences set forth in any of SEQ ID NOs: 352-392; and/or
said eukaryotic cells are totipotent stem cells;
14. The method or genetically modified cell of claim 13 .

A DNA editing agent for use in therapy to treat a disease in a subject in need thereof, said agent being silenced according to the method of any one of claims 1-6 , 9-13 , or 21 . producing an RNA molecule having silencing activity and/or specificity, said RNA molecule having silencing activity against transcripts of genes associated with the onset or progression of said disease, thereby rendering said subject How to treat.

A method of introducing silencing activity into a first RNA molecule in a cell, comprising:
(a)
i. the first nucleic acid sequence is transcribed into said first RNA molecule within the cell;
ii. a third RNA molecule wherein the sequence of said first RNA molecule has partial homology, except for sequence identity, with the sequence of said second RNA molecule, said second RNA molecule having silencing activity; processable into an RNA molecule, said second RNA molecule being encoded by a second nucleic acid sequence within said cell; and iii. said first RNA molecule is either unprocessable or processable differently than the second RNA molecule such that the first RNA molecule has the same property of silencing activity as the third RNA molecule. is not processed into an RNA molecule with
selecting said first nucleic acid sequence in said cell;
(b) modifying said first nucleic acid sequence to encode a modified first RNA molecule, wherein said modified first RNA molecule is processable into a fourth RNA molecule as well as being processable into a third RNA molecule, such that the fourth RNA molecule has a silencing activity of the same nature as the third RNA molecule;
thereby introducing a silencing activity into said first RNA molecule;
method including.

said second RNA molecule is an RNA molecule having a secondary structure that allows it to be processed into an RNA having silencing activity, optionally wherein said silencing activity is mediated through association with RISC to be
optionally,
(i) said RNA molecule having a secondary structure that allows it to be processed into an RNA having silencing activity is microRNA (miRNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA) or URNA), small nucleolar RNA (snoRNA), small cajal RNA (scaRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), repeat-derived RNA, autonomous and non-autonomous transposable and selected from the group consisting of RNAs from retrotransposable elements, RNAs of autonomous and non-autonomous transposable and retrotransposable elements, and long non-coding RNAs (lncRNAs); or
(ii) the first nucleic acid sequence provides a secondary structure that allows the modified first RNA molecule to be processed into a fourth RNA molecule;
optionally, modifying said first nucleic acid sequence is such that the modified first RNA molecule has essentially the same secondary structure as the second RNA molecule, optionally the secondary structure of the second RNA molecule or
The first nucleic acid molecule is a human (H. sapiens)-derived gene selected from the group consisting of genes having a sequence set forth in any one of SEQ ID NOS: 352 to 392,
24. The method of claim 23 .