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-12065669-B2
Publication Date
2024-08-20
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. The polypeptide comprises a reverse transcriptase domain and an endonuclease domain, and comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1988. The system further includes 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.
Dependent embodiments further define the 5′ UTR and 3′ UTR by identity relationships to SEQ ID NO: 1986 and SEQ ID NO: 1987, respectively, and include optional regulatory and localization elements. Additional content describes insertion-length constraints, an NLS sequence, and delivery in lipid nanoparticles (LNPs).
The disclosed system is associated with lipid nanoparticle delivery of the DNA-modifying system. The partial content further describes Gene Writer compositions, lipid nanoparticle formulations, and delivery-related features including surface ligands such as GalNAc/ASGPR and SORT molecules.
Claims Coverage
The consolidated claim coverage includes one independent DNA-modifying system claim. The inventive features focus on the reverse transcriptase and endonuclease domain-containing polypeptide with a sequence identity threshold, the template RNA architecture with polypeptide-binding 5′ and 3′ UTRs plus a heterologous object sequence, and delivery as an LNP.
Dual-domain DNA-modifying polypeptide with SEQ ID NO: 1988 identity
A polypeptide or nucleic acid encoding the polypeptide comprising a reverse transcriptase domain and an endonuclease domain, and comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1988.
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 defined by SEQ ID NO with identity threshold
A 5′ UTR containing a sequence selected from SEQ ID NO: 1986 or a sequence having at least 95% identity to SEQ ID NO: 1986.
3′ UTR defined by SEQ ID NO with identity threshold
A 3′ UTR containing a sequence selected from SEQ ID NO: 1987 or a sequence having at least 95% identity to SEQ ID NO: 1987.
Insertion length thresholds for heterologous object insertion
The insertion is at least 45 nucleotides long, with possible minimum lengths selected from 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides.
NLS sequence specified by amino acid sequence
An NLS sequence having the amino acid sequence PKKKRKV (SEQ ID NO: 2409).
Delivery via a lipid nanoparticle
A lipid nanoparticle comprising the system of claim 1.
Claim coverage centers on a DNA-modifying system defined by a reverse transcriptase and endonuclease domain-containing polypeptide with at least 95% identity to SEQ ID NO: 1988 and a template RNA with polypeptide-binding 5′ and 3′ UTR sequences plus a heterologous object sequence, with additional constraints on specific UTR sequences, insertion-length thresholds, an NLS sequence, and delivery in an LNP.
Stated Advantages
Improved sequence-specific insertion of longer edits compared to CRISPR or Cre/loxP.
Increasing plant fitness traits, including yield and vigor.
Providing drought, heat, cold, salt, and metal tolerance.
Reducing allergen production.
Targeted delivery can be enhanced via ligand-displayed LNPs, including GalNAc/ASGPR targeting.
Minimizing reactive impurities such as aldehydes can reduce RNA damage.
Documented Applications
Delivering a Gene Writer composition to lung/pulmonary cells using lipid nanoparticle formulations.
Gene Writer systems for genome editing and plant modification.
Plant trait modification for increased yield, vigor, drought, heat, cold, salt, and metal tolerance, and reduced allergen production.
Mammalian-cell and in vivo contexts for retrotransposase-based Gene Writing, including targeted integration and nucleotide substitution.
Delivery to liver/hepatocytes using lipid nanoparticles containing labeled lipid formulas (i)-(xv).
Delivery to lung/pulmonary cells using lipid nanoparticles containing labeled lipid formulas (i)-(xv).
Ligand-displayed LNP targeting using GalNAc/ASGPR targeting to enhance targeted delivery to specific tissues/cells.
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