Compositions and methods for gene editing by targeting transferrin
Inventors
BROOKS, Alan Richard • VO, Karen
Assignees
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Publication Number
US-12215320-B2
Publication Date
2025-02-04
Expiration Date
Abstract
Provided include compositions, methods, and systems for modulating the expression, function, and/or activity of a target gene, for example a blood-clotting protein such as Factor VIII (FVIII), in a cell by genome editing. Also provided include compositions, methods, and systems for treating a subject having or suspected of having a disorder or health condition, e.g., Hemophilia A, employing ex vivo and/or in vivo genome editing.
Core Innovation
The invention provides genome-editing systems that target intron 1 of an endogenous transferrin gene in a cell. The system includes a DNA endonuclease or nucleic acid encoding the DNA endonuclease, a guide RNA with a spacer sequence complementary to a sequence within intron 1 of the endogenous transferrin gene, and a donor template comprising a nucleic acid sequence encoding a protein-of-interest, wherein the protein-of-interest is Factor VIII (FVIII).
The disclosed approach directs donor-mediated integration at the transferrin locus to support endogenous-promoter-driven expression. The donor template architecture comprises splice acceptor, POI, polyA, and homology arms, and transferrin promoter/signal peptide splicing features are used so that secretion is provided with an authentic N-terminus. The protein-of-interest can be implemented as FVIII with FVIII-BDD, and codon-optimized donors are described in connection with splice-acceptor replacement of the signal peptide.
The disclosure also includes constraints intended to reduce CpG di-nucleotide content in the protein-of-interest coding sequence, including thresholds down to none. Editing components and formats include transferrin-intron-1 gRNAs/spacers, DNA endonuclease-gRNA RNP precomplexing, and delivery formats including liposome or lipid nanoparticle systems and AAV donor templates.
Claims Coverage
The independent claims cover a genome-editing system with three core inventive elements: a DNA endonuclease or nucleic acid encoding it, a gRNA targeting intron 1 of an endogenous transferrin gene, and a donor template encoding FVIII. The claim set further includes five refinements covering gRNA spacer selection, POI CpG content, RNP formation, formulation, and donor template delivery format.
Transferrin intron 1 targeted genome-editing system encoding FVIII
A system comprising a DNA endonuclease or nucleic acid encoding the DNA endonuclease; a guide RNA comprising a spacer sequence complementary to a sequence within intron 1 of an endogenous transferrin gene in a cell; and a donor template comprising a nucleic acid sequence encoding a protein-of-interest, wherein the protein-of-interest is Factor VIII.
Specific transferrin intron 1 gRNA spacer sets with limited mismatches
The gRNA comprises one of several defined spacer sequences or variants thereof with no more than 3 mismatches.
Low-CpG FVIII coding sequence constraints
The nucleic acid sequence encoding the protein-of-interest contains about or less than 20 CpG di-nucleotides, or about or less than 10 CpG di-nucleotides, or about or less than 5 CpG di-nucleotides, or contains no CpG di-nucleotides.
DNA endonuclease precomplexed with gRNA as an RNP complex
A DNA endonuclease is precomplexed with gRNA to form a ribonucleoprotein complex.
Liposome or lipid nanoparticle formulation of the endonuclease
The DNA endonuclease or nucleic acid encoding the DNA endonuclease is formulated within a liposome or lipid nanoparticle.
AAV-vector encoded donor template
The donor template is encoded in an Adeno Associated Virus vector.
Overall claim coverage centers on a transferrin intron 1 targeting CRISPR/Cas genome-editing system delivering a donor template encoding FVIII, with dependent limitations specifying particular gRNA spacer sets and mismatch constraints, constraining CpG di-nucleotide content of the FVIII coding sequence, requiring DNA endonuclease/gRNA RNP precomplexing, and defining endonuclease formulation and AAV-encoded donor delivery.
Stated Advantages
Targeted integration at a transferrin intron 1 target site with resulting Factor VIII expression levels reported in Hemophilia A and NSG mice.
Reported assessment of on-target and in vivo relevance, including on-target activity in primary human hepatocytes.
Off-target profiling is reported using GUIDE-seq, including characterization of off-target sites for specific transferrin gRNAs.
Integration into transferrin intron 1 is reported to give substantially higher FVIII expression than albumin intron 1 when normalized to integration frequency.
Documented Applications
In vivo genome editing in mouse liver for Hemophilia A and NSG mice, including targeted integration and FVIII expression evaluation.
On-target evaluation in primary human hepatocytes for transferrin intron 1 targeting with Factor VIII donor templates.
Off-target profiling of transferrin gRNAs using GUIDE-seq to characterize off-target sites and reported off-target frequencies.
In vitro assessment in HepG2 cells using GUIDE-seq and TIDE analysis, including off-target profiling for transferrin intron 1-targeting guides.
In vivo comparison in Hemophilia A mice of FVIII cassette integration into transferrin intron 1 versus albumin intron 1, reported using integration frequencies and normalized FVIII activity/expression.
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