COMPOSITIONS AND METHODS FOR BASE EDITING A PHENYLALANINE HYDROXYLASE POLYNUCLEOTIDE
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.
1 . 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.
2 . The method of claim 1 , wherein the alteration to the nucleobase in codon 408 of the PAH polynucleotide results in a W408R amino acid alteration in a PAH polypeptide encoded by the PAH polynucleotide.
3 . The method of claim 1 , wherein the base editor comprises an amino acid sequence having at least 85% sequence identity to a sequence selected from those listed in Table 2.
4 . The method of claim 1 , wherein the guide RNA comprises a spacer comprising at least 10 contiguous nucleotides of a spacer sequence selected from those listed in Table 1.
5 . The method of claim 1 , wherein the one or more polynucleotides encoding the base editor comprise an RNA sequence encoding a polypeptide having at least 85% sequence identity to an amino acid sequence selected from those listed in Table 2.
6 . 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.
7 . The method of claim 6 , wherein the alteration to the nucleobase in codon 408 of the PAH polynucleotide results in a W408R amino acid alteration in a PAH polypeptide encoded by the PAH polynucleotide.
8 . The method of claim 6 , wherein the base editor comprises an amino acid sequence having at least 85% sequence identity to a sequence selected from those listed in Table 2.
9 . The method of claim 6 , wherein the guide RNA comprises a spacer comprising at least 10 contiguous nucleotides of a spacer sequence selected from those listed in Table 1.
10 . The method of claim 6 , wherein the one or more polynucleotides encoding the base editor comprise an RNA sequence encoding a polypeptide having at least 85% sequence identity to an amino acid sequence selected from those listed in Table 2.
11 . A modified cell comprising an alteration in a nucleobase of an PAH polynucleotide, wherein the alteration is prepared by the method of claim 1 , and wherein the alteration increases activity of the encoded PAH polypeptide as compared to a control cell without the alteration.
12 . 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.
13 . The base editor system of claim 12 , wherein the alteration to the nucleobase in codon 408 of the PAH polynucleotide results in a W408R amino acid alteration in a PAH polypeptide encoded by the PAH polynucleotide.
14 . The base editor system of claim 12 , wherein the base editor comprises an amino acid sequence having at least 85% sequence identity to a sequence selected from those listed in Table 2.
15 . The base editor system of claim 12 , wherein the guide RNA comprises a spacer comprising at least 10 contiguous nucleotides of a spacer sequence selected from those listed in Table 1.
16 . The base editor system of claim 12 , wherein the one or more polynucleotides encoding the base editor comprise an RNA sequence encoding a polypeptide having at least 85% sequence identity to an amino acid sequence selected from those listed in Table 2.
17 . A set of polynucleotides encoding the base editor system of claim 12 , or a component thereof.
18 . A lipid nanoparticle comprising the base editor system of claim 12 .
19 . A pharmaceutical composition comprising the base editor system of claim 12 , and a pharmaceutically acceptable excipient.
20 . A kit comprising the base editor system comprising the base editor system of claim 12 , wherein the kit further comprises a container.
21 . A guide RNA comprising a sequence listed in Table 1.