Methods and compositions for modulating a genome

Patent metadata

Field	Value
Publication number	US-12398392-B2
Application number	US-202418623612-A
Country	US
Kind code	B2
Filing date	Apr 1, 2024
Priority date	Aug 28, 2018
Publication date	Aug 26, 2025
Grant date	Aug 26, 2025

Abstract

Methods and compositions for modulating a target genome are disclosed. The composition may comprise a first RNA encoding a polypeptide comprising a retrotransposase reverse transcriptase domain and a retrotransposase endonuclease domain. The composition may also comprise a second RNA comprising a sequence that binds the polypeptide and a heterologous object sequence. The composition may insert the sequence of the heterologous object sequence into a target DNA.

First claim

What is claimed is:

1. A method of modifying a target DNA strand in a cell, tissue or subject, the method comprising administering a composition for modifying DNA to the cell, tissue or subject, thereby modifying the target DNA strand;
wherein the composition for modifying DNA comprises:
(a) a first engineered RNA encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain and (ii) an endonuclease domain, wherein the polypeptide comprises the amino acid sequence SEQ ID NO: 1387, or a sequence having at least 99% identity thereto; and
(b) a second engineered RNA comprising a template RNA, wherein the template RNA comprises (i) the sequence of SEQ ID NO: 1142, or a sequence having at least 99% identity thereto, that binds the encoded polypeptide, and (ii) a heterologous object sequence; and
wherein (a) and (b) are separate RNAs.

2. The method of claim 1 , wherein the template RNA further comprises a sequence comprising at least 20 nucleotides of at least 80% identity to a target DNA.

3. The method of claim 1 , wherein the template RNA comprises:
(iii) at least 3 bases of identity to a target DNA at the 3′ end of the template RNA, and (iv) at least 3 bases of identity to the target DNA at the 5′ end of the template RNA.

4. The method of claim 1 , wherein the heterologous object sequence encodes an enzyme, a membrane protein, a blood factor, an intracellular protein, an extracellular protein, a structural protein, a signaling protein, a regulatory protein, a transport protein, or a motor protein.

5. The method of claim 1 , wherein the polypeptide further comprises one or both of a nuclear localization signal and a nucleolar localization signal.

6. The method of claim 1 , wherein the composition comprises only RNA, or comprises more RNA than DNA by an RNA:DNA ratio of at least 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.

7. The method of claim 1 , wherein the template RNA does not encode a reverse transcriptase or an endonuclease.

8. The method of claim 1 , wherein the template RNA comprises: at least 10 bases of identity to a target DNA at the 3′ end of the template RNA, and (iv) at least 10 bases of identity to the target DNA at the 5′ end of the template RNA.

9. The method of claim 1 , wherein the template RNA further comprises the sequence of SEQ ID NO: 1265, or a sequence having at least 95% identity thereto.

10. The method of claim 1 , wherein the template RNA further comprises the sequence of SEQ ID NO: 1265, or a sequence having at least 99% identity thereto.

11. The method of claim 1 , wherein the template RNA further comprises the sequence of SEQ ID NO: 1265.

12. The method of claim 1 , wherein the template RNA further comprises the sequence of SEQ ID NO: 1142.

13. The method of claim 1 , wherein the target DNA is a genomic safe harbor (GSH) site.

14. The method of claim 1 , wherein the heterologous object sequence encodes a therapeutic polypeptide or fragment thereof.

15. The method of claim 1 , wherein the heterologous object sequence comprises a non-coding sequence or a regulatory sequence.

16. The method of claim 1 , wherein the composition comprises no more than 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, or 0.01% DNA by mass or by molar amount of nucleic acid.

17. The method of claim 1 , wherein the composition is capable of modifying DNA in the absence of homologous recombination activity.

18. The method of claim 15 , wherein the regulatory sequence is a promoter.

Assignees

• Flagship Pioneering Innovations Vi Llc

Inventors

• Rubens Jacob Rosenblum
• Von Maltzahn Geoffrey A
• Citorik Robert James
• Steinberg Barrett Ethan
• Li Donghui
• Salomon William Edward

Classifications

• C12N2800/00

  Nucleic acids vectors · CPC title

• C12N15/907

  in mammalian cells · CPC title

• C12N15/85

  for animal cells · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title