Methods and compositions for modulating a genome
Inventors
CLEAVER, Stephen Hoyt • Steinberg, Barrett Ethan • Rubens, Jacob Rosenblum • Citorik, Robert James • Salomon, William Edward • WANG, Zi Jun
Assignees
Flagship Pioneering Inc • Flagship Pioneering Innovations VI Inc
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Publication Number
US-12037602-B2
Publication Date
2024-07-16
Expiration Date
Abstract
Methods and compositions for modulating a target genome are disclosed.
Core Innovation
The invention relates to a system for modifying DNA that includes a polypeptide, or a nucleic acid encoding the polypeptide, where the polypeptide comprises a reverse transcriptase domain and an endonuclease domain and includes an amino acid sequence at least 95% identical to SEQ ID NO: 1947. The system further includes a template RNA that binds the polypeptide through a 5′ UTR sequence and a 3′ UTR sequence and includes a heterologous object sequence.
The disclosure further describes dependent refinements that specify the 5′ UTR and 3′ UTR sequences by reference to SEQ ID NO: 1945 and SEQ ID NO: 1946, respectively, or sequences having at least 95% identity to them. Additional refinements specify an NLS sequence including PKKKRKV, insertion length thresholds, and delivery via a lipid nanoparticle.
The disclosure also describes thermostable family-B DNA polymerases that include a reverse transcriptase domain and a 3′-5′ exonuclease domain having DnaQ-like motifs, together with conserved RT signatures, ExoI/ExoII/ExoIII motifs, a YX(3)D pattern, DEDDy-type features, beta hairpin structure, and active site switching. It further describes functional metrics for DNA-binding, RNA-binding, nicking/endonuclease activity, reverse transcriptase initiation stringency, completion/termination, and integration fidelity, and references evolved variants generated by PACE/PANCE.
Claims Coverage
The independent claim coverage centers on a DNA-modifying system defined by a dual-domain polypeptide and a template RNA that binds the polypeptide via specified UTR regions and carries a heterologous object sequence. Dependent features further specify UTR sequence constraints, an NLS sequence, insertion length thresholds, and a lipid nanoparticle delivery vehicle.
Reverse transcriptase and endonuclease polypeptide with sequence identity to SEQ ID NO: 1947
A polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises a reverse transcriptase domain and an endonuclease domain and has an amino acid sequence at least 95% identical to the sequence of SEQ ID NO: 1947.
Template RNA with 5′ and 3′ UTR binding regions and heterologous object sequence
A template RNA comprising a 5′ UTR sequence that binds the polypeptide, a 3′ UTR sequence that binds the polypeptide, and a heterologous object sequence.
5′ UTR sequence constraint
The 5′ UTR includes a sequence matching SEQ ID NO: 1945 or a sequence having at least 95% identity to it.
3′ UTR sequence constraint
The 3′ UTR contains a sequence identical to SEQ ID NO: 1946 or a sequence having at least 95% identity to it.
Defined NLS sequence
An NLS sequence characterized by the amino acid sequence PKKKRKV (SEQ ID NO: 2409).
Minimum insertion length threshold in nucleotides
The system is configured such that its insertion is at least a specified minimum length in nucleotides, including 45 through 100 nucleotides.
Lipid nanoparticle comprising the DNA modifying system
A lipid nanoparticle comprising the DNA modifying system.
Overall, the claim coverage is anchored on a dual-domain DNA-modifying polypeptide with a sequence identity requirement to SEQ ID NO: 1947 and a template RNA that binds the polypeptide through 5′ and 3′ UTR regions while carrying a heterologous object sequence. The dependent features further specify UTR sequence requirements, an NLS sequence, insertion length thresholds, and delivery via a lipid nanoparticle.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Delivering GeneWriter compositions to targeted cell types, including liver and/or hepatocytes, and lung or pulmonary cells including lung endothelial cells.
Gene writing and targeted DNA integration evaluation in human cells, including integration increases assessed relative to non-gRNA controls using GFP expression and ddPCR-based copy-number measurement at an AAVS1 genomic site.
Genetic modification example involving a nucleotide substitution in SERPINA1 (alpha-1 antitrypsin deficiency) using a reverse transcription template and a Cas9 nickase with a corresponding target design.
Downstream formulation and delivery examples using lipid nanoparticles containing modified firefly luciferase mRNA, with reported luciferase expression including ranked liver delivery for specific lipid formulations.
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