JPWO2021178717A5

JPWO2021178717A5 -

Info

Publication number: JPWO2021178717A5
Application number: JP2022552817A
Authority: JP
Publication date: 2024-03-13

Description

特許ファイル又は出願ファイルは、色付きで仕上げた少なくとも１つの図面を含有する。色付き図面を有する特許公報又は特許出願公報のコピーは、請求と必須の料金の支払いがあれば直ぐに、庁によって提供される。
本発明の実施形態において、例えば以下の項目が提供される。
（項目１）
ＤＮＡを修飾するためのシステムであって、
（ａ）ポリペプチド又はポリペプチドをコードする核酸（例えば、ＤＮＡ又はｍＲＮＡ）であって、前記ポリペプチドが（ｉ）逆転写酵素ドメインと（ｉｉ）エンドヌクレアーゼドメインとを含み、（ｉ）又は（ｉｉ）の一方又は両方が、表３Ｂ、表１０、又は表１１のエレメントの核酸配列によってコードされるアミノ酸配列又はそれと少なくとも７０％、７５％、８０％、８５％、９０％、９５％、９６％、９７％、９８％、又は９９％の同一性を有する配列を有する、ポリペプチド又は核酸；及び
（ｂ）（ｉ）前記ポリペプチドに結合する配列と（ｉｉ）異種目標配列とを含む鋳型ＲＮＡ（又は前記鋳型ＲＮＡをコードするＤＮＡ）
を含むシステム。
（項目２）
ＤＮＡを修飾するためのシステムであって、
（ａ）ポリペプチド又はポリペプチドをコードする核酸（例えば、ＤＮＡ又はｍＲＮＡ）であって、前記ポリペプチドが（ｉ）逆転写酵素ドメインと（ｉｉ）エンドヌクレアーゼドメインとを含み、（ｉ）又は（ｉｉ）の一方又は両方が、表３Ｂ、表１０、又は表１１のエレメントの核酸配列によってコードされるアミノ酸配列又はそれと１０、２０、３０、４０、５０、６０、７０、８０、９０、又は１００ヌクレオチドの差異を超えない配列を有する、ポリペプチド又は核酸；及び
（ｂ）（ｉ）前記ポリペプチドに結合する配列と（ｉｉ）異種目標配列とを含む鋳型ＲＮＡ（又は前記鋳型ＲＮＡをコードするＤＮＡ）
を含むシステム。
（項目３）
ＤＮＡを修飾するためのシステムであって、
（ａ）ポリペプチド又はポリペプチドをコードする核酸（例えば、ＤＮＡ又はｍＲＮＡ）であって、前記ポリペプチドが（ｉ）逆転写酵素ドメインと（ｉｉ）標的ＤＮＡ結合ドメインとを含み、（ｉ）又は（ｉｉ）の一方又は両方が、表３Ｂ、表１０、又は表１１のエレメントの核酸配列によってコードされるアミノ酸配列又はそれと少なくとも７０％、７５％、８０％、８５％、９０％、９５％、９６％、９７％、９８％、又は９９％の同一性を有する配列を有する、ポリペプチド又は核酸；及び
（ｂ）（ｉ）前記ポリペプチドに結合する配列と（ｉｉ）異種目標配列とを含む鋳型ＲＮＡ（又は前記鋳型ＲＮＡをコードするＤＮＡ）
を含むシステム。
（項目４）
ＤＮＡを修飾するためのシステムであって、
（ａ）ポリペプチド又は前記ポリペプチドをコードする核酸であって、前記ポリペプチドが（ｉ）逆転写酵素（ＲＴ）ドメインと、（ｉｉ）ＤＮＡ結合ドメイン（ＤＢＤ）と；（ｉｉｉ）エンドヌクレアーゼドメイン、例えばニッカーゼドメインとを含む、ポリペプチド又は核酸；及び
（ｂ）（例えば、５’から３’に）（ｉ）任意選択で、標的部位（例えば、標的ゲノム中の部位の編集されない鎖）に結合する配列と、（ｉｉ）任意選択で、前記ポリペプチドに結合する配列と、（ｉｉｉ）異種目標配列と、（ｉｖ）３’相同ドメインとを含む鋳型ＲＮＡ（又は前記鋳型ＲＮＡをコードするＤＮＡ）
を含み、
前記ＲＴドメインが、表３Ｂ、表１０、又は表１１の配列、又はそれと少なくとも７０％、７５％、８０％、８５％、９０％、９５％、９６％、９７％、９８％、又は９９％の同一性を有する配列を有する、システム。 A patent or application file contains at least one drawing executed in color. Copies of patent publications or patent application publications with color drawings will be provided by the Office upon request and payment of the required fee.
In the embodiment of the present invention, the following items are provided, for example.
(Item 1)
A system for modifying DNA, the system comprising:
(a) a polypeptide or a nucleic acid (e.g., DNA or mRNA) encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain and (ii) an endonuclease domain; one or both of ii) is at least 70%, 75%, 80%, 85%, 90%, 95%, 96 %, 97%, 98%, or 99% identity; and
(b) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds to the polypeptide and (ii) a heterologous target sequence;
system containing.
(Item 2)
A system for modifying DNA, the system comprising:
(a) a polypeptide or a nucleic acid (e.g., DNA or mRNA) encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain and (ii) an endonuclease domain; one or both of ii) is an amino acid sequence encoded by or 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid sequences encoded by the nucleic acid sequences of the elements of Table 3B, Table 10, or Table 11; polypeptides or nucleic acids having sequences that differ by no more than nucleotides; and
(b) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds to the polypeptide and (ii) a heterologous target sequence;
system containing.
(Item 3)
A system for modifying DNA, the system comprising:
(a) a polypeptide or a nucleic acid (e.g., DNA or mRNA) encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain and (ii) a target DNA binding domain; (ii) at least 70%, 75%, 80%, 85%, 90%, 95% of, or the amino acid sequence encoded by the nucleic acid sequence of an element of Table 3B, Table 10, or Table 11; polypeptides or nucleic acids having sequences with 96%, 97%, 98%, or 99% identity; and
(b) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds to the polypeptide and (ii) a heterologous target sequence;
system containing.
(Item 4)
A system for modifying DNA, the system comprising:
(a) a polypeptide or a nucleic acid encoding said polypeptide, said polypeptide comprising: (i) a reverse transcriptase (RT) domain; (ii) a DNA binding domain (DBD); (iii) an endonuclease domain. , a polypeptide or nucleic acid comprising, for example, a nickase domain; and
(b) (e.g., 5' to 3') (i) optionally a sequence that binds to a target site (e.g., the non-edited strand of the site in the target genome); A template RNA (or DNA encoding said template RNA) comprising a sequence that binds to a peptide, (iii) a heterologous target sequence, and (iv) a 3' homology domain.
including;
the RT domain is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence of Table 3B, Table 10, or Table 11; A system having sequences having an identity of .

Claims

A system for modifying DNA, the system comprising:
a) a polypeptide or a nucleic acid encoding said polypeptide, said polypeptide comprising (i) a reverse transcriptase domain and (ii) an endonuclease domain, said polypeptide having at least the sequence of SEQ ID NO: 1988; with a polypeptide or nucleic acid comprising an amino acid sequence with 95% identity;
b) (i) a 5'UTR sequence that binds to said polypeptide;
(ii) a 3'UTR sequence that binds to the polypeptide; and
(ii) Heterogeneous objective array
template RNA containing
system, including.

2. The system of claim 1, wherein the 5'UTR comprises the sequence SEQ ID NO: 1986 or a sequence having at least 95% identity thereto.

3. The system of claim 1 or 2, wherein the 3'UTR comprises the sequence SEQ ID NO: 1987 or a sequence having at least 95% identity thereto.

A system for modifying DNA, the system comprising:
(a) a polypeptide or a nucleic acid encoding said polypeptide, said polypeptide comprising (i) a reverse transcriptase domain and (ii) an endonuclease domain, said polypeptide having the sequence of SEQ ID NO: 1947; with a polypeptide or nucleic acid comprising an amino acid sequence having at least 95% identity;
(b) (i) a 5'UTR sequence that binds to the polypeptide;
(ii) a 3'UTR sequence that binds to the polypeptide; and
(ii) Heterogeneous objective array
template RNA containing
system, including.

5. The system of claim 4, wherein the 5'UTR comprises the sequence SEQ ID NO: 1945 or a sequence having at least 95% identity thereto.

5. The system of claim 4, wherein the 5'UTR comprises the sequence SEQ ID NO: 1945.

7. The system of any one of claims 4 to 6, wherein the 3'UTR comprises the sequence SEQ ID NO: 194 or a sequence having at least 95% identity thereto.

7. The system of any one of claims 4-6, wherein the 3'UTR comprises the sequence SEQ ID NO: 1946.

9. The system of any one of claims 1 to 8, wherein the heterologous sequence of interest encodes a therapeutic or human polypeptide or a fragment or variant thereof.

A system according to any one of claims 1 to 8, wherein the heterologous sequence of interest encodes a chimeric antigen receptor (CAR).

A system according to any one of claims 1 to 8, wherein the heterogeneous target sequence comprises a control sequence.

The control sequence is
(a) is a promoter;
(b) is an enhancer;
(c) is a binding site for an endogenous regulatory component;
(d) is an miRNA binding site, or
(e) altering the expression of an endogenous gene or non-coding RNA;
The system according to claim 11.

7. The system of any of the preceding claims, wherein the polypeptide comprises a nuclear localization signal (NLS).

14. The system of claim 13, wherein the NLS is fused to the N-terminus of the polypeptide.

14. The system of claim 13, wherein the NLS is fused to the C-terminus of the polypeptide.

14. The system of claim 13, wherein the NLS sequence comprises the amino acid sequence of PKKKRKV (SEQ ID NO: 2409).

17. The system of any one of claims 1-16, wherein the polypeptide comprises a linker having an amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 1023).

The system according to any one of claims 1 to 16, wherein the polypeptide comprises a linker having the amino acid sequence of GGGS (SEQ ID NO: 1024).

19. The system of claim 17 or 18, wherein the linker is placed between the NLS and the remainder of the polypeptide.

The template RNA is
(a) Poly A site;
(b) regulatory elements of woodchuck hepatitis virus (WPRE); and/or
(c) Kozak sequence
A system according to any of the preceding claims, comprising:

6. The system of any of the preceding claims, wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are two separate nucleic acids.

7. The system of any of the preceding claims, wherein (a) comprises RNA encoding the polypeptide and (b) comprises template RNA.

7. The system of any of the preceding claims, wherein the nucleic acid encoding the polypeptide comprises a coding sequence that is codon-optimized for expression in human cells.

A system according to any of the preceding claims, capable of inducing additions, deletions or modifications of protein coding sequences to the genome of mammalian cells.

A system according to any of the preceding claims, capable of inducing the addition, deletion or modification of non-coding RNA to the genome of a mammalian cell.

26. The system of claim 24 or 25, wherein the insertion is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length.

The mammalian cell is
(a) human cells;
(b) primary cells; and/or
(b) T cells
21. The system of claim 20.

An in vitro or ex vivo method of modifying a target DNA strand in a cell, tissue or subject, comprising administering to said cell, tissue or subject a system according to any of the preceding claims, said system comprising: A method of reverse transcribing a template RNA sequence into said target DNA strand, thereby modifying said target DNA strand.

Lipid nanoparticles (LNPs) comprising a system according to any one of claims 1 to 27.

A system according to any one of claims 1 to 27 for use in a method for modifying a target DNA strand in a cell, tissue or subject.