Compositions and methods for base editing a phenylalanine hydroxylase polynucleotide
AssigneeBeam Therapeutics Inc.
InventorsTanggis Bohnuud, Mandy Sze Lee, Yi Yu
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Abstract
Compositions and methods for treating phenylketonuria by introducing one or more alterations into a phenylalanine hydroxylase (PAH) polynucleotide in a cell. In particular embodiments, the disclosure provides a base editor system (e.g., a fusion protein or complex comprising a programable DNA binding protein, a nucleobase editor, and gRNA) for modifying a PAH polynucleotide, where the alteration is associated with an increase in the level or activity of a PAH polypeptide encoded by the polynucleotide.
Claims21
A method of editing a nucleobase of an phenylalanine hydroxylase (PAH) polynucleotide, the method comprising contacting the PAH polynucleotide with a guide RNA, or a polynucleotide encoding the guide RNA, and a base editor comprising a fusion protein or a protein complex comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, wherein said guide RNA targets said base editor to effect an alteration to a nucleobase in codon 408 of the PAH polynucleotide, wherein the deaminase domain comprises or consists of the following amino acid sequence with one or more amino acid alterations MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:
- 1), or a fragment thereof lacking only the N-terminal methionine, wherein the one or more amino acid alterations comprises or consists of a set of alterations selected from the group consisting of:
- a) I76Y, V82T, Y123H, Y147R, and Q154R;
- b) I76Y, V82T, Y123H, Y147R, F149Y, and Q154R;
- and
- c) I76Y, V82T, Y123H, Y147D, F149Y, Q154R, T166I, and D167N.
A method of treating phenylketonuria (PKU) in a subject in need thereof, the method comprising administering to a cell of the subject a base editor system comprising a base editor comprising a fusion protein or protein complex comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, and a guide RNA that targets the base editor to effect an alteration to a nucleobase in codon 408 of a phenylalanine hydroxylase (PAH) polynucleotide in the cell, or a polynucleotide encoding the guide RNA, thereby treating hypertension in the subject, wherein the deaminase domain comprises or consists of the following amino acid sequence with one or more amino acid alterations MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:
- 1), or a fragment thereof lacking only the N-terminal methionine, wherein the one or more amino acid alterations comprises or consists of a set of alterations selected from the group consisting of:
- a) I76Y, V82T, Y123H, Y147R, and Q154R;
- b) I76Y, V82T, Y123H, Y147R, F149Y, and Q154R;
- and
- c) I76Y, V82T, Y123H, Y147D, F149Y, Q154R, T166I, and D167N.
A base editor system comprising a base editor or one or more polynucleotides encoding the base editor, wherein the base editor comprises a nucleic acid programmable DNA binding protein domain (napDNAbp) and a deaminase domain, and a guide RNA that targets said base editor to effect an alteration to a nucleobase in codon 408 of a PAH polynucleotide, wherein the deaminase domain comprises or consists of the following amino acid sequence with one or more amino acid alterations MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:
- 1), or a fragment thereof lacking only the N-terminal methionine, wherein the one or more amino acid alterations comprises or consists of a set of alterations selected from the group consisting of:
- a) I76Y, V82T, Y123H, Y147R, and Q154R;
- b) I76Y, V82T, Y123H, Y147R, F149Y, and Q154R;
- and
- c) I76Y, V82T, Y123H, Y147D, F149Y, Q154R, T166I, and D167N.
A guide RNA comprising a sequence listed in Table 1.
Dependencies are inferred from the claim text.