Methods and compositions for modulating a genome
Inventors
Rubens, Jacob Rosenblum • VON MALTZAHN, GEOFFREY A. • Citorik, Robert James • Steinberg, Barrett Ethan • Li, Donghui • Salomon, William Edward
Assignees
Flagship Pioneering Innovations VI Inc • Tessera Therapeutics Inc
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Publication Number
US-12398392-B2
Publication Date
2025-08-26
Expiration Date
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.
Core Innovation
The invention describes retrotransposon-based genome editing and DNA-modifying systems for inserting a heterologous object sequence into a target DNA site. The system includes a polypeptide comprising a reverse transcriptase domain and an endonuclease domain, optionally with a separate DNA-binding domain, and a template RNA comprising a sequence that binds the polypeptide plus the heterologous object sequence to be reverse-transcribed and inserted.
The document states that the target DNA strand is modified in a cell, tissue, or subject by administering a DNA-modifying composition comprising separate engineered RNAs. A first engineered RNA encodes a polypeptide with a reverse transcriptase domain and an endonuclease domain, and a second engineered template RNA includes a sequence that binds the encoded polypeptide as well as the heterologous object sequence.
The invention further characterizes modular non-LTR retrotransposon components, including APE-type and RLE-type retrotransposons, for longer-sequence insertions and for insertion at endogenous loci such as rDNA and genomic safe harbor sites. System architectures described include separate versus fused RNAs/DNA arrangements and specific localization, promoter, and orientation configurations, with supporting analyses of integration junctions, copy-number and integration fidelity, and reduced DNA damage and tumor-suppressor responses.
Claims Coverage
The relevant claim family covers a method of modifying a target DNA strand in a cell, tissue, or subject by administering a composition for modifying DNA that uses two separate engineered RNAs: one encoding a reverse transcriptase/endonuclase polypeptide and one template RNA that binds the encoded polypeptide and carries the heterologous object sequence. The inventive features are the engineered polypeptide domains and sequence identity, the template RNA binding sequence and heterologous object sequence, and the use of separate RNAs.
Separate engineered RNAs for target DNA modification
A composition for modifying DNA comprises a first engineered RNA encoding a polypeptide with a reverse transcriptase domain and an endonuclease domain, and a second engineered RNA comprising a template RNA that binds the encoded polypeptide and includes a heterologous object sequence, wherein the RNAs are separate.
Reverse transcriptase and endonuclease polypeptide encoded by first engineered RNA
The first engineered RNA encodes a polypeptide that comprises a reverse transcriptase domain and an endonuclease domain, the polypeptide comprising the amino acid sequence SEQ ID NO: 1387 or a sequence having at least 99% identity thereto.
Template RNA that binds encoded polypeptide and includes heterologous object sequence
The second engineered RNA comprises a template RNA that includes a sequence of SEQ ID NO: 1142 or a sequence having at least 99% identity thereto that binds the encoded polypeptide, and includes a heterologous object sequence for reverse transcription and insertion.
Across the claim coverage, DNA modification is achieved by administering a DNA-modifying composition that uses separate engineered RNAs: one RNA encoding a SEQ ID NO: 1387 reverse transcriptase/endonuclase polypeptide and a second template RNA containing a SEQ ID NO: 1142-binding sequence plus a heterologous object sequence.
Stated Advantages
Reduced DNA damage and reduced tumor-suppressor responses.
Documented Applications
Modifying a target DNA strand in a cell, tissue, or subject by administering the DNA-modifying composition.
Inserting heterologous object sequences into endogenous genomic loci, including an rDNA locus and genomic safe harbor sites.
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