Methods and compositions for modulating a genome
Inventors
Bothmer, Anne Helen • Cotta-Ramusino, Cecilia Giovanna Silvia • Salomon, William Edward • Rubens, Jacob Rosenblum • Citorik, Robert James • WANG, Zi Jun • Kim, Kyusik • KOTLAR, Randi Michelle • RAY, Ananya • ALTSHULER, Robert Charles • Kumar, Sandeep • Roquet, Nathaniel • Steinberg, Barrett Ethan
Assignees
Flagship Pioneering Inc • Flagship Pioneering Innovations VI Inc • Tessera Therapeutics Inc
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Publication Number
US-12157898-B2
Publication Date
2024-12-03
Expiration Date
Abstract
Methods and compositions for modulating a target genome are disclosed. This disclosure relates to novel compositions, systems and methods for altering a genome at one or more locations in a host cell, tissue or subject, in vivo or in vitro. In particular, the invention features compositions, systems and methods for inserting, altering, or deleting sequences of interest in a host genome.
Core Innovation
The invention relates to a fusion protein comprising a reverse transcriptase (RT) domain having the amino acid sequence of SEQ ID NO: 3225 and a Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain. The fusion protein includes a peptide linker between the RT domain and the Cas9 nickase, and can include an NLS fused to the N-terminus of the Cas9 nickase domain.
The Cas9 nickase domain is refined by sequence identity and selectable variant embodiments, including SpyCas9 (N863A), together with a functional activity threshold relative to a comparable Cas9 nickase molecule not fused to an RT domain. The disclosure also places the fusion protein within GENE WRITER™ system architectures that use RT-domain and Cas-domain arrangements.
The broader disclosure describes GENE WRITER™ genome editing configurations that use a first and a second DNA strand nick, with inward and outward nick orientations relative to PAM sites and binding sites. Additional disclosed systems include template RNA/gRNA architecture for target-primed reverse transcription, DNA-free all-RNA implementations, and lipid nanoparticle delivery.
Claims Coverage
The consolidated claim coverage centers on one independent inventive structure: a fusion protein comprising an RT domain having the amino acid sequence of SEQ ID NO: 3225 and a Cas9 nickase domain, with the RT domain C-terminal of the Cas9 nickase domain. The claim coverage is further refined by dependent features including linker length, NLS placement, Cas9 nickase sequence identity or variant selection, and a quantitative activity threshold.
Reverse transcriptase and Cas9 nickase fusion with specified domain order
A fusion protein comprising an RT domain having the amino acid sequence of SEQ ID NO: 3225 and a Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain.
Peptide linker between RT and Cas9 nickase
The fusion protein includes a peptide linker between the RT domain and Cas9 nickase, with the peptide linker length constrained to 2 to 40 amino acids.
NLS fused to the Cas9 nickase N-terminus
The fusion protein includes an NLS fused to the N-terminus of the Cas9 nickase domain.
Cas9 nickase domain defined by sequence identity
The Cas9 nickase domain contains an amino acid sequence with at least 99% identity to SEQ ID NO: 3269.
SpyCas9 (N863A) nickase domain
The fusion protein includes a SpyCas9 (N863A) nickase domain.
Cas9 nickase activity threshold relative to an unfused reference
The Cas9 nickase domain has activity at least 50% of that of a comparable Cas9 nickase molecule not fused to an RT domain.
Overall, the claim coverage focuses on an RT–Cas9 nickase fusion with RT domain placement at the C-terminus of the Cas9 nickase, further constrained by inter-domain linker length, NLS positioning, selected Cas9 nickase variant and sequence identity, and retention of Cas9 nickase activity at a quantified threshold.
Stated Advantages
Low indel increase during simultaneous editing.
Outward nicking reduces DSB risk and undesired insertions and deletions (indels).
Improved specificity.
Longer sequence integration versus existing approaches.
Tissue and cell targeting for delivery to liver hepatocytes and lung endothelial/pulmonary cells.
Minimizing reactive impurities (aldehydes) that can damage RNA and impair reverse transcription.
Documented Applications
Multiplex GENE WRITER™ editing of two loci (HEK3 and HBB) using template RNA and second-nick gRNAs.
DNA-free all-RNA implementations delivering Cas9-RT mRNA plus Template RNA and optional second-nick gRNA into HEK293T and primary human CD4+ T cells.
Delivery using lipid nanoparticles (LNP) formulated with selected ionizable lipids, reporting RNA encapsulation and luciferase expression in primary hepatocytes and mouse liver.
Large genomic insertions using an R2 retrotransposase-derived RT template payload (GFP) and retrotransposase-based integration selection assays.
Therapeutic applications are discussed, including broad use in connection with transgenes and listed disease gene targets.
Applications involving CAR-T, CAR-NK, and CAR-NKT are discussed in the context of the disclosed system.
Genome editing of host genomic DNA for inserting, substituting, or deleting sequences using the described RT/template RNA target-primed reverse transcription framework.
Delivery of a GeneWriter system to liver and hepatocytes using LNP formulations.
Delivery of a GeneWriter system to lung endothelial cells and pulmonary cells using LNP formulations.
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