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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
  • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
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  • C12N15/102—Mutagenizing nucleic acids
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
  • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/22—Endodeoxyribonucleases producing 3'-phosphomonoesters (3.1.22)

Definitions

• Bxbl recombinase also known as Bxbl integrase, is an LSR encoded by phage Bxbl that facilitates integration of the phage DNA into the Mycobacterium smegmatis genome (Russell et al., Biotechniques (2016) 40(4): doi.org/10.2144/000112150).
• the recombinase contains an N-terminal catalytic domain similar to that of smaller resolvases/invertases and a larger C-terminal domain responsible for coordination of activities unique to LSRs.
• the numbers underneath the protein structure in (A) indicate the approximate distance, from the N-terminus of the protein: of the C-terminal border of the NTD (—150 amino acids), the N-terminal border of the CTD (300 amino acids), and the C-terminus of the CTD (600 amino acids).
• FIG. 5 is a schematic illustrating the chromosomal recombination assay in human K562 cells.
• Bxbl facilitates the targeted integration (H) of a donor plasmid into a chromosomal Bxbl attB pseudo-site (an endogenous human sequence with some homology to the natural Bxbl attB target site).
• White and dark gray boxes indicate attB and attP sites, respectively.
• F-Primer and R-Primer refer to primer binding locations used for a PCR-based NGS assay to quantify TI events. Shown at bottom is the product resulting from a successful TI event.
• Sequence alterations at these sites that match alterations in Table 1 are shown in the second, third, and fourth column, where the second column shows the relevant position in the target site of the alteration, the third column shows the alterations at the given position in the left half-site of the target site listed in the first column, and the fourth column shows the alteration in the target site at the relevant position of the right half-site. If the base at the indicated position of the indicated half-site matches the target preferences of WT Bxbl (e.g. A at position 10, C at position 9, or A at position 7), then the base is indicated as “WT Bxbl.”
• WT Bxbl target preferences
• the term “functionally analogous sequence” refers to a sequence of amino acid residues or a protein domain having the same or substantially the same biological function.
• the RD domain may approximately correspond to amino acids 140-287 (numbering according to SEQ ID NO: 1) in wildtype Bxbl recombinase or the functionally analogous sequence in Bxbl recombinase variants, or the functionally analogous sequence in orthologous serine recombinase(s) and their variants.
• G318 to I, K, R, or W;
• the Bxbl variant RD domain may comprise any one or more of the following amino acid mutations:
• F147 to A, K, or R;
• NLRPLGS SEQ ID NO: 78
• the system comprises a mixture of different Bxbl recombinase variants and/or other orthologous serine recombinase variants, and/or the nucleic acids that encode them, as well as any associated donor DNA molecules.
• the attB sequences described herein comprise any one of the sequences of SEQ ID NOs: 83-130, as shown in Table 3. In some embodiments, the attB sequences described herein comprise any one of the sequences of SEQ ID NOs: 4-40, as shown in Table 4. In some embodiments, the attP sequences described herein comprise any one of the sequences of SEQ ID NOs: 41-76, as shown in Table 5. In some embodiments, the attB or attP sequences may comprise a sequence selected from SEQ ID NOs: 80-82 and 134-136.
• the expression vector is an AAV viral vector and is introduced to the target human cell by a recombinant AAV virion whose genome comprises the construct, including having the AAV Inverted Terminal Repeat (ITR) sequences on both ends to allow the production of the AAV virion in a production system such as an insect cell/baculovirus production system or a mammalian cell production system.
• the AAV may be engineered such that its capsid proteins have reduced immunogenicity or enhanced transduction ability in humans.
• Viral vectors described herein may be produced using methods known in the art. Any suitable permissive or packaging cell type may be employed to produce the viral particles.
• mammalian (e.g., 293) or insect (e.g., sf9) cells may be used as the packaging cell line.
• the systems can be used to modify pluripotent stem cells prior to their differentiation into multiple cell types.
• a lymphoid cell precursor may be modified prior to differentiation into lymphoid cell types such as regulatory T cells, effector T cells, natural killer cells, etc.
• Systems of the present disclosure comprising more than one Bxbl recombinase variant or ortholog ous recombinase variant, in particular, can be used to prepare cells with multiple integrated, excised, or inverted genes at once, including pluripotent cells.
• systems containing more than one of the provided recombinase variants may be used to prepare, e.g., allogeneic T cells.
• any method for introduction of proteins or nucleic acid molecules to a plant cell is also contemplated, such as Agrobacterium tumefaciens-meAia eA T- DNA delivery.
• the methods of the present disclosure are used to insert or excise a gene or regulatory sequence associated with a disease towards restoring normal gene expression or activity.
• the methods of the present disclosure may target a particular allele of a gene, e.g., a wild-type or mutated allele.
• the allele may be associated with cancer.
• the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

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• 2023
  • 2023-01-27 EP EP23706980.2A patent/EP4469569A1/en active Pending
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