Fah-modulating compositions and methods
AssigneeFlagship Pioneering Innovations VI, LLC
InventorsAnne Helen Bothmer, Luciano Henrique Apponi, Aamir Mir, Zhan Wang
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Abstract
The disclosure provides, e.g., compositions, systems, and methods for targeting, editing, modifying, or manipulating a host cell's genome at one or more locations in a DNA sequence in a cell, tissue, or subject. Gene modifying systems for treating hereditary tyrosinemia type 1 (HT1) disease are described.
Claims72
A template RNA comprising, from 5′ to 3′:
- (i) a gRNA spacer that is complementary to a first portion of the human FAH gene, wherein the gRNA spacer has a sequence comprising the core nucleotides of a gRNA spacer sequence of Table 1A, Table 1B, or Table 1C, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the gRNA spacer;
- (ii) a gRNA scaffold that binds a gene modifying polypeptide (e.g., binds the Cas domain of the gene modifying polypeptide),
- (iii) a heterologous object sequence comprising a mutation region to introduce a mutation into a second portion of the human FAH gene (wherein optionally the heterologous object sequence comprises, from 5′ to 3′, a post-edit homology region, a mutation region, and a pre-edit homology region), and
- (iv) a primer binding site (PBS) sequence comprising at least 5, 6, 7, or 8 bases with 100% identity to a third portion of the human FAH gene.
A template RNA comprising, from 5′ to 3′:
- (i) a gRNA spacer that is complementary to a first portion of the human FAH gene,
- (ii) a gRNA scaffold that binds a gene modifying polypeptide (e.g., binds the Cas domain of the gene modifying polypeptide),
- (iii) a heterologous object sequence comprising a mutation region to introduce a mutation into a second portion of the human FAH gene, wherein the heterologous object sequence comprises the core nucleotides of an RT template sequence of Table 3A, Table 3B, or Table 3C, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence;
- and
- (iv) a PBS sequence comprising at least 5, 6, 7, or 8 bases of 100% identity to a third portion of the human FAH gene.
The template RNA or gene modifying system of any one of the preceding claims, wherein the gRNA spacer comprises about 1, 2, 3, or more flanking nucleotides of the gRNA spacer.
The template RNA or gene modifying system of any one of the preceding claims, wherein the heterologous object sequence comprises about 2, 3, 4, 5, 10, 20, 30, 40, or more flanking nucleotides of the RT template sequence.
The template RNA or gene modifying system of any one of the preceding claims, wherein the heterologous object sequence comprises between about 8-30, 9-25, 10-20, 11-16, or 12-15 (e.g., about 11-16) nucleotides.
The template RNA or gene modifying system of any one of the preceding claims, wherein the mutation region comprises 1, 2, or 3 nucleotide positions of sequence difference relative to the corresponding portion of the human FAH gene.
The template RNA or gene modifying system of any one of the preceding claims wherein the mutation region comprises at least 2 nucleotide positions of sequence difference relative to the corresponding portion of the human FAH gene.
The template RNA or gene modifying system, of any one of the preceding claims, wherein the post-edit homology region and/or pre-edit homology region comprises 100% identity to the FAH gene.
The template RNA or gene modifying system of any one of the preceding claims, wherein the PBS sequence additionally comprises about 1, 2, 3, 4, 5, 6, 7, or more flanking nucleotides.
The template RNA or gene modifying system of any one of the preceding claims, wherein the PBS sequence comprises about 5-20, 8-16, 8-14, 8-13, 9-13, 9-12, or 10-12 (e.g., about 9-12) nucleotides.
The template RNA or gene modifying system of any one of the preceding claims, wherein the PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in the FAH gene.
The gene modifying system of any one of the preceding claims, wherein the domains of the gene modifying polypeptide are joined by a peptide linker.
The gene modifying system of any one of the preceding claims, wherein the gene modifying polypeptide further comprise one or more nuclear localization sequences (NLS).
A template RNA comprising a sequence of a template RNA of Table 4A, Table 4B, or Table 4C, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
A template RNA comprising a sequence of a template RNA of Table 4A, Table 4B, or Table 4C.
A gene modifying system comprising:
- (iii) a template RNA comprising a sequence of a template RNA of Table 4A, Table 4B, or Table 4C, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto;
- and
- (iv) a second-nick gRNA sequence from the same row of Table 4A, Table 4B, or Table 4C as (i), a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
A gene modifying system comprising:
- (iii) a template RNA comprising a sequence of a template RNA of Table 4;
- and
- (iv) a second-nick gRNA sequence from the same row of Table 4A, Table 4B, or Table 4C as (i).
A host cell (e.g., a mammalian cell, e.g., a human cell) comprising the template RNA or gene modifying system of any one of the preceding claims.
The gene modifying system or method of any one of the preceding claims, wherein introduction of the system into a target cell results in a correction of a pathogenic mutation in the FAH gene.
The gene modifying system or method of any one of the preceding claims, wherein the pathogenic mutation is a W262X, IVS12+5G>A, and/or IVS6−1G>Cmutation, and wherein the correction comprises an amino acid substitution of X262W, IVS12+5A>G, and/or IVS6−1C>G.
The gene modifying system or method of any one of the preceding claims, wherein introduction of the system into a target cell results in a mutation that causes the restoration of the function of the FAH gene.
The gene modifying system or method of any of the preceding claims, wherein correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, or more) of target nucleic acids.
The gene modifying system or method of any of the preceding claims, wherein correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, or more) of target cells.
The method of any of the preceding claims, wherein the cell is a mammalian cell, such as a human cell.
The method of any one of the preceding claims, wherein the subject is a human.
The method of any of the preceding claims, wherein the contacting occurs ex vivo, e.g., wherein the cell's or subject's DNA is modified ex vivo.
The method of any of the preceding claims, wherein the contacting occurs in vivo, e.g., wherein the cell's or subject's DNA is modified in vivo.
The method of any of the preceding claims, wherein contacting the cell or the subject with the system comprises contacting the cell or a cell within the subject with a nucleic acid (e.g., DNA or RNA) encoding the gene modifying polypeptide under conditions that allow for production of the gene modifying polypeptide.
Dependencies are inferred from the claim text.