Compositions and methods for altering complement activation
AssigneeBeam Therapeutics Inc.
InventorsLei Johnson, Tanggis Bohnuud, Lo-I Cheng, Michael Packer, Brian Cafferty
Life of this patent
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Abstract
Compositions and methods for reducing complement activation by introducing one or more alterations into a complement factor B (CFB) polynucleotide in a cell. In particular embodiments, the invention of the disclosure features a base editor system (e.g., a fusion protein or complex comprising a programmable DNA binding protein, a nucleobase editor, and gRNA) for modifying a CFB polynucleotide, where the modification is associated with reduced expression, and/or reduced activity of the CFB polypeptide encoded by the polynucleotide.
Claims21
A method of altering a nucleobase of a complement factor B (CFB) polynucleotide, the method comprising contacting the CFB polynucleotide with a base editor system comprising one or more guide polynucleotides, or one or more polynucleotides encoding the one or more guide polynucleotides, and a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotide encoding the base editor, wherein:
- (a) said one or more guide polynucleotides targets said base editor to effect an alteration of a nucleobase of the CFB polynucleotide that:
- i. disrupts a splice site in the CFB polynucleotide,
- ii. alters a start codon in the CFB polynucleotide,
- iii. alters a TATA box in the CFB polynucleotide, and/or
- iv. introduces a new stop codon in the CFB polynucleotide;
- (b) the deaminase domain comprises a TadA variant (TadA*) comprising an amino acid sequence having at least 90% sequence identity to the following TadA*7.10 amino acid sequence:
- MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE IMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:
- 1), or a fragment thereof lacking only the N-terminal methionine, wherein the TadA* further comprises a combination of amino acid alterations compared to the TadA*7.10 amino acid sequence selected from the group consisting of:
- i. 176Y, V82T, Y123H, Y147T, and Q154S,
- ii. Y123H, Y147R, and Q154R,
- iii. I76Y, Y133H, Y147R, and Q154R,
- iv. V82S, and Q164R,
- v. I76Y, V82S, Y123H, Y147R, and Q154R, and
- vi. I76Y, V82T, Y123H, Y147R, and Q154R;
- and/or
- (c) the one or more guide polynucleotides comprises a nucleic acid sequence selected from CCUCAGAUGUCUAUGUGUUU (SEQ ID NO:
- 1524), UGCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1535), TUUGCUCCCCAUGGCGTUUGGA (SEQ ID NO:
- 3476), CCCCAUGGCGUUGGAAGGCA (SEQ ID NO:
- 3443), and UGCUCCCCAUGGCGUUGGAA (SEQ ID NO:
- 3467) and/or comprising at least 10-23 contiguous nucleotides of a spacer nucleic acid sequence listed in any one of Tables 2A to 2F;
- thereby altering the nucleobase of the CFB polynucleotide.
A method of altering a nucleobase of a complement factor B (CFB) polynucleotide, the method comprising contacting the CFB polynucleotide with one or more guide polynucleotides, or one or more polynucleotides encoding the one or more guide polynucleotides, and a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, wherein:
- (a) the deaminase domain comprises a cytidine deaminase or a TadA variant (TadA*) comprising an amino acid sequence having at least 90% sequence identity to the following TadA*7.10 amino acid sequence:
- MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:
- 1), or a fragment thereof lacking only the N-terminal methionine, wherein the TadA* further comprises a combination of amino acid alterations compared to the TadA*7.10 amino acid sequence selected from the group consisting of:
- i. 176Y, V82T, Y123H, Y147T, and Q154S,
- ii. Y123H, Y147R, and Q154R,
- iii. I76Y, Y133H, Y147R, and Q154R,
- iv. V82S, and Q164R,
- v. I76Y, V82S, Y123H, Y147R, and Q154R, and
- vi. I76Y, V82T, Y123H, Y147R, and Q154R;
- and
- (b) the one or more guide polynucleotides comprise a spacer comprising a nucleotide sequence selected from the group consisting of CCUCAGAUGUCUAUGUGUUU (SEQ ID NO:
- 1524;
- TSBTx3826), UGCUCCCCAUGGCGUUGGAA (SEQ ID NO:
- 3467;
- gRNA3657), UUGCUCCCCAUGGCGUUGGA (SEQ ID NO:
- 3476;
- gRNA3658), CCCCAUGGCGUUGGAAGGCA (SEQ ID NO:
- 3443;
- gRNA3660), GCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1534;
- TSBTx3837), UGCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1535;
- TSBTx3837), and UCUCACCUCUGCAAGUAUUG (SEQ ID NO:
- 1529;
- TSBTx3835);
- thereby altering the nucleobase of the CFB polynucleotide.
A cell produced by the method of claim 7.
A base editor system comprising a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, and one or more guide polynucleotides comprising at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of a spacer comprising a nucleotide sequence selected from CCUCAGAUGUCUAUGUGUUU (SEQ ID NO:
- 1524), UGCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1535), UUGCUCCCCAUGGCGUUGGA (SEQ ID NO:
- 3476), CCCCAUGGCGUUGGAAGGCA (SEQ ID NO:
- 3443), and UGCUCCCCAUGGCGUUGGAA (SEQ ID NO:
- 3467) and/or listed in Table 2A to 2F, or one or more polynucleotides encoding the one or more guide polynucleotides.
A guide polynucleotide comprising a sequence listed in any one of Tables 1A to 2F.
A base editor system comprising a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, and one or more guide polynucleotides, or one or more polynucleotides encoding the one or more guide polynucleotides wherein:
- (a) the deaminase domain is a cytidine deaminase or comprises a TadA variant (TadA*) comprising an amino acid sequence having at least 90% sequence identity to the following TadA*7.10 amino acid sequence:
- MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE IMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:
- 1), wherein the TadA* further comprises a combination of amino acid alterations compared to the TadA*7.10 amino acid sequence selected from the group consisting of:
- i. 176Y, V82T, Y123H, Y147T, and Q154S,
- ii. Y123H, Y147R, and Q154R,
- iii. I76Y, Y133H, Y147R, and Q154R,
- iv. V82S, and Q164R,
- v. I76Y, V82S, Y123H, Y147R, and Q154R, and
- vi. I76Y, V82T, Y123H, Y147R, and Q154R;
- (b) the one or more guide polynucleotides comprise a spacer comprising a nucleotide sequence selected from the group consisting of:
- CCUCAGAUGUCUAUGUGUUU (SEQ ID NO:
- 1524;
- TSBTx3826), UGCUCCCCAUGGCGUUGGAA (SEQ ID NO:
- 3467;
- gRNA3657), UUGCUCCCCAUGGCGUUGGA (SEQ ID NO:
- 3476;
- gRNA3658), CCCCAUGGCGUUGGAAGGCA (SEQ ID NO:
- 3443;
- gRNA3660), GCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1534;
- TSBTx3837), UGCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1535;
- TSBTx3837), and UCUCACCUCUGCAAGUAUUG (SEQ ID NO:
- 1529;
- TSBTx3835);
- (c) the one or more guide polynucleotides comprises a sequence selected from the group consisting of:
- End-mod SpCas9 guide polynucleotide
- (SEQ ID NO:
- 440)
- mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
- AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsmUsmUsmU,
- HM01:
- (SEQ ID NO:
- 440)
- mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGU
- UAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmG
- mAmGmUmCmGmGmUmGmCmUsmUsmUsmU,
- and
- NLS (bpsv40):
- (SEQ ID NOs:
- 440 and 446)
- mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU
- AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsmU-NHC6-CrossL-
- ac-CKRTADGSEFESPKKKRKV,
- wherein “N” represents any nucleotide, “mN” indicates a 2′-OMe modification of the nucleotide “N”, and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate (PS), and wherein the number of N nucleotides is between and 25;
- and
- (d) the napDNAbp is an spCas9 nickase polypeptide that binds a protospacer adjacent motif (PAM) selected from the group consisting of NGA, NGC, and NGG, wherein “N” is any nucleotide.
A composition comprising:
- a) a polynucleotide encoding a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, wherein the deaminase domain is a cytidine deaminase or comprises a TadA variant (TadA*) comprising an amino acid sequence having at least 90% sequence identity to the following TadA*7.10 amino acid sequence:
- MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:
- 1), wherein the TadA* further comprises a combination of amino acid alterations compared to the TadA*7.10 amino acid sequence selected from the group consisting of:
- i. 176Y, V82T, Y123H, Y147T, and Q154S,
- ii. Y123H, Y147R, and Q154R,
- iii. I76Y, Y133H, Y147R, and Q154R,
- iv. V82S, and Q164R,
- v. I76Y, V82S, Y123H, Y147R, and Q154R, and
- vi. I76Y, V82T, Y123H, Y147R, and Q154R;
- and
- b) a guide RNA comprises a spacer comprising a nucleotide sequence selected from the group consisting of CCUCAGAUGUCUAUGUGUUU (SEQ ID NO:
- 1524;
- TSBTx3826), UGCUCCCCAUGGCGUUGGAA (SEQ ID NO:
- 3467;
- gRNA3657), UUGCUCCCCAUGGCGUUGGA (SEQ ID NO:
- 3476;
- gRNA3658), CCCCAUGGCGUUGGAAGGCA (SEQ ID NO:
- 3443;
- gRNA3660), GCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1534;
- TSBTx3837), UGCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1535;
- TSBTx3837), and UCUCACCUCUGCAAGUAUUG (SEQ ID NO:
- 1529;
- TSBTx3835) and/or comprising at least 10-23 contiguous nucleotides of a spacer nucleic acid sequence listed in any one of Tables 2A to 2F.
A lipid nanoparticle (LNP) composition comprising:
- a) an mRNA encoding a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, wherein the deaminase domain is a cytidine deaminase or comprises a TadA variant (TadA*) comprising an amino acid sequence having at least 90% sequence identity to the following TadA*7.10 amino acid sequence:
- MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:
- 1), wherein the TadA* further comprises a combination of amino acid alterations compared to the TadA*7.10 amino acid sequence selected from the group consisting of:
- i. I76Y, V82T, Y123H, Y147T, and Q154S,
- ii. Y123H, Y147R, and Q154R,
- iii. I76Y, Y133H, Y147R, and Q154R,
- iv. V82S, and Q164R,
- v. I76Y, V82S, Y123H, Y147R, and Q154R, and
- vi. I76Y, V82T, Y123H, Y147R, and Q154R;
- and
- b) a guide RNA comprises a spacer comprising a nucleotide sequence selected from the group consisting of CCUCAGAUGUCUAUGUGUUU (SEQ ID NO:
- 1524;
- TSBTx3826), UGCUCCCCAUGGCGUUGGAA (SEQ ID NO:
- 3467;
- gRNA3657), UUGCUCCCCAUGGCGUUGGA (SEQ ID NO:
- 3476;
- gRNA3658), CCCCAUGGCGUUGGAAGGCA (SEQ ID NO:
- 3443;
- gRNA3660), GCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1534;
- TSBTx3837), UGCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1535;
- TSBTx3837), and UCUCACCUCUGCAAGUAUUG (SEQ ID NO:
- 1529;
- TSBTx3835) and/or comprising at least 10-23 contiguous nucleotides of a spacer nucleic acid sequence listed in any one of Tables 2A to 2F.
A method of treatment comprising administering to a subject a lipid nanoparticle (LNP) compositing comprising:
- a) an mRNA encoding a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, wherein the deaminase domain is a cytidine deaminase or comprises a TadA variant (TadA*) comprising an amino acid sequence having at least 90% sequence identity to the following TadA*7.10 amino acid sequence:
- MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO:
- 1), wherein the TadA* further comprises a combination of amino acid alterations compared to the TadA*7.10 amino acid sequence selected from the group consisting of:
- i. 176Y, V82T, Y123H, Y147T, and Q154S,
- ii. Y123H, Y147R, and Q154R,
- iii. I76Y, Y133H, Y147R, and Q154R,
- iv. V82S, and Q164R,
- v. I76Y, V82S, Y123H, Y147R, and Q154R, and
- vi. I76Y, V82T, Y123H, Y147R, and Q154R;
- and
- b) a guide RNA comprises a spacer comprising a nucleotide sequence selected from the group consisting of CCUCAGAUGUCUAUGUGUUU (SEQ ID NO:
- 1524;
- TSBTx3826), UGCUCCCCAUGGCGUUGGAA (SEQ ID NO:
- 3467;
- gRNA3657), UUGCUCCCCAUGGCGUUGGA (SEQ ID NO:
- 3476;
- gRNA3658), CCCCAUGGCGUUGGAAGGCA (SEQ ID NO:
- 3443;
- gRNA3660), GCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1534;
- TSBTx3837), UGCUUACAAUGACUGAGAUCU (SEQ ID NO:
- 1535;
- TSBTx3837), and UCUCACCUCUGCAAGUAUUG (SEQ ID NO:
- 1529;
- TSBTx3835) and/or comprising at least 10-23 contiguous nucleotides of a spacer nucleic acid sequence listed in any one of Tables 2A to 2F.
Dependencies are inferred from the claim text.