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-12031129-B2
Publication Date
2024-07-09
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 relates to gene writer/gene writing retrotransposon-based genome editing/modulating systems and a composition for modifying DNA comprising a first engineered RNA and a second engineered RNA, where the two RNAs are separate. The first engineered RNA encodes a polypeptide that comprises a reverse transcriptase domain and an endonuclease domain, and the second engineered RNA comprises a template RNA that binds the encoded polypeptide and comprises a heterologous object sequence.
The disclosure describes a two-part engineered nucleic acid architecture using a polypeptide or an RNA encoding it, optionally combined with a DNA-binding domain derived from non-LTR retrotransposons such as APE-type or RLE-type clades. The template RNA includes a sequence that binds the encoded polypeptide and a heterologous object payload sequence to be inserted into target DNA, including endogenous loci such as a genomic safe harbor, for example an rDNA locus.
The GENE WRITER™ genome editing approach uses thermostable retrotransposase-derived genome editor polypeptides and engineered two-part RNA components, including a template RNA and a reverse transcriptase/endonuclease polypeptide subunit. The document emphasizes activity at 37°C and reports insertion site contexts such as 28S rDNA variants, 18S rDNA, 5S rDNA, tRNA, and U2 snRNA.
Claims Coverage
The independent claim coverage centers on one composition for modifying DNA and identifies two inventive features: a first engineered RNA encoding a reverse transcriptase and endonuclease polypeptide, and a second separate engineered RNA comprising a template RNA that binds the polypeptide and carries a heterologous object sequence.
Two separate engineered RNAs for DNA modification
A composition for modifying DNA comprising a first engineered RNA and a second engineered RNA, wherein the two RNAs are separate.
Polypeptide with reverse transcriptase and endonuclease domains
The first engineered RNA encodes a polypeptide comprising a reverse transcriptase domain and an endonuclease domain, including amino acid sequences according to any of SEQ ID NOs: 1016-1021.
Template RNA that binds the encoded polypeptide and carries a heterologous object sequence
The second engineered RNA comprises a template RNA that binds the encoded polypeptide and comprises a heterologous object sequence, including a sequence according to SEQ ID NO: 1140.
Overall, the claims require a two-part engineered RNA system for DNA modification: a first RNA encoding a reverse transcriptase/endonuclease polypeptide and a second separate RNA providing a template RNA that binds the polypeptide and carries a heterologous object sequence. Dependent claim refinements further characterize localization signals, nucleic-acid composition constraints, template identity requirements, and functional narrowing conditions, and specify that the heterologous object sequence can be a therapeutic polypeptide or fragment.
Stated Advantages
Insertion of payloads without detectable double-strand breaks.
Reduced DNA repair/tumor suppressor upregulation.
Thermostable activity at 37°C.
Reduced truncation behavior and scarless integration behavior.
Documented Applications
Modifying DNA by inserting a heterologous object sequence into target DNA, including endogenous loci such as a genomic safe harbor or rDNA locus.
Genome editing / modifying DNA using the GENE WRITER™ system with insertion into genomic contexts such as 28S rDNA variants, 18S rDNA, 5S rDNA, tRNA, and U2 snRNA.
Encoding and inserting heterologous object sequences, including therapeutic polypeptides or fragments thereof.
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