METHODS AND COMPOSITIONS FOR TREATING ALPHA-1 ANTITRYPSIN DEFICIENCY
Compositions and methods for editing deleterious mutations associated with Alpha-1 Antitrypsin Deficiency (A1AD). In particular embodiments, the invention provides methods for treating A1AD using a modified adenosine base editor with improved on-target editing and decreased off-target editing to correct mutations associated with A1AD.
1 . A nucleic acid encoding a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain, wherein the napDNAbp domain comprises an alteration selected from the group consisting of M1135L, E1250K, A1283D, Q1136Y, R1337K, R765A, and Q768A of an amino acid sequence, or a fragment thereof lacking an N-terminal methionine, that is at least 90% identical to:
(SEQ ID NO: 554)
MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA
LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR
LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD
LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP
INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP
NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
LLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPEKYKEI
FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPY
YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD
LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ
LKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD
SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV
MGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP
VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD
SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL
TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI
REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK
YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI
TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
QTGGFSKESILPKGNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVE
KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK
YSLFELENGRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPE
DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
PIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQ
SITGLYETRIDLSQLGGD.
2 . The nucleic acid of claim 1 , wherein the napDNAbp domain comprises a combination of alterations referenced to SEQ ID NO: 554 selected from the group consisting of:
a R1337K;
b. Q1136Y and R1337K;
C. M1135L, Q1136Y, and R1337K;
d. M1135L, Q1136Y, A1283D, and R1337K;
e M1135L, E1250K, A1283D, and R1337K;
f. Q1136Y, A1283D, and E1250K;
g. M1135L, Q1136Y, A1283D, and R1337K; and
h. R765A, Q768A, M1135L, Q1136Y, A1283D, and R1337K.
3 . The nucleic acid of claim 1 , wherein the adenosine deaminase domain comprises an alteration at an amino acid position selected from the group consisting of L36, I76, V82, Y147, Q154, and N157 of an amino acid sequence, or a fragment thereof lacking an N-terminal methionine, that is at least 85% identical to:
(SEQ ID NO: 1)
MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG
LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG
RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR
MPRQVFNAQKKAQSSTD.
4 . The nucleic acid of claim 3 , wherein the adenosine deaminase domain comprises an amino acid alteration selected from the group consisting of L36H, 176Y, V82T, Y147T, Q154S, and N157K referenced to SEQ ID NO: 1.
5 . The nucleic acid sequence of claim 4 , wherein the adenosine deaminase domain comprises the amino acid alterations L36H, I76Y, V82T, Y147T, Q154S, and N157K referenced to SEQ ID NO: 1
6 . The nucleic acid of claim 5 , wherein the adenosine deaminase domain comprises an amino acid sequence that is at least 90% identical to the following amino acid sequence:
(SEQ ID NO: 426)
SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVHNNRVIGEGWNRAIGL
HDPTAHAEIMALRQGGLVMQNYRLYDATLYTTFEPCVMCAGAMIHSRIGR
VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRM
PRSVFKAQKKAQSSTD.
7 . The nucleic acid of claim 1 , wherein the adenosine deaminase domain comprises the amino acid sequence of SEQ ID NO: 426.
8 . The nucleic acid of claim 1 , further comprising a linker between the adenosine deaminase and the napDNAbp.
9 . The nucleic acid of claim 8 , wherein the linker comprises an amino acid sequence selected from the group consisting of: SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 357), EGGSEEEEESGS (SEQ ID NO: 542), and KGPKPKKEESEK (SEQ ID NO: 439).
10 . The nucleic acid of claim 1 , further comprising a nuclear localization sequence (NLS).
11 . The nucleic acid of claim 10 , wherein the NLS comprises the amino acid sequence
(SEQ ID NO: 438)
EGADKRTADGSEFESPKKKRKV.
12 . The nucleic acid of claim 1 , wherein the base editor comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 440-450.
13 . The nucleic acid of claim 12 , wherein the base editor comprises any one of SEQ ID NOs: 440-450.
14 . The nucleic acid of claim 1 , wherein the nucleic acid comprises RNA.
15 . The nucleic acid of claim 14 , wherein the nucleic acid is an mRNA molecule.
16 . A base editor system comprising a nucleic acid encoding a base editor comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain and (ii) a guide polynucleotide,
wherein the napDNAbp domain comprises an alteration selected from the group consisting of M1135L, E1250K, A1283D, Q1136Y, R1337K, R765A, and Q768A of an amino acid sequence, or a fragment thereof lacking an N-terminal methionine, that is at least 90% identical to:
(SEQ ID NO: 554)
MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT
RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD
EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI
QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL
TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT
EITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF
YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLK
DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT
NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK
VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV
LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDF
LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV
DELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL
QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD
NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV
VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN
GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNS
DKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP
IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKGNELALPSKYVNFLYLAS
HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI
REQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQ
LGGD.
17 . The base editor system of claim 16 , wherein the guide polynucleotide is a single guide RNA (sgRNA).
18 . The base editor system of claim 17 , wherein the sgRNA comprises a spacer comprising a nucleotide sequence selected from the group consisting of: 5′-ACCAUCGACAAGAAAGGGACUGA-3′ (SEQ ID NO: 466); 5′-CCAUCGACAAGAAAGGGACUGA-3′ (SEQ ID NO: 559); 5′-CAUCGACAAGAAAGGGACUGA-3′ (SEQ ID NO: 560); 5′-AUCGACAAGAAAGGGACUGA-3′ (SEQ ID NO: 561); 5′-UCGACAAGAAAGGGACUGA-3′ (SEQ ID NO: 562); and 5′-CGACAAGAAAGGGACUGA-3′ (SEQ ID NO: 563).
19 . The base editor system of claim 17 , wherein the sgRNA comprises a nucleotide sequence selected from the group consisting of:
(SEQ ID NO: 558)
5′-ACCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC
AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU
GGCACCGAGU CGGUGCUUUU-3′;
(SEQ ID NO: 564)
5′-CCAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC
AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU
GGCACCGAGU CGGUGCUUUU-3′;
(SEQ ID NO: 565)
5′-CAUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC
AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU
GGCACCGAGU CGGUGCUUUU-3′;
(SEQ ID NO: 566)
5′-AUCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC
AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU
GGCACCGAGU CGGUGCUUUU-3′;
(SEQ ID NO: 567)
5′-UCGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC
AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU
GGCACCGAGU CGGUGCUUUU-3′;
and
(SEQ ID NO: 568)
5′-CGACAAGAAAGGGACUGA GUUUUAGAGC UAGAAAUAGC
AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU
GGCACCGAGU CGGUGCUUUU-3′.
20 . The base editor system of claim 19 , wherein the sgRNA comprises a nucleotide having a 2′-OMe modification, a 2′-fluoro (F) modification, and/or a phosphorothioate modification.
21 . A guide RNA comprising a nucleotide sequence, from 5′ to 3′, selected from the group consisting of:
(SEQ ID NO: 569)
mAsmUsmCsGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGU
UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU
GCmUsmUsmUsU;
(SEQ ID NO: 570)
mAsmUsmCsGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGU
UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU
GCUsmUsmUsmU,
wherein the guide is covalently linked at the 3′ end to a peptide with the amino acid sequence CKRTADGSEFESPKKKRKV (SEQ ID NO: 543);
(SEQ ID NO: 571)
mAsmUsmCsmGmAmCmAmAmGmAfAfAfGIGGsAsfCfUGsmAmGUsUsUsfUfAmGmAmGm
CmUmAmGmAmAmAmUmAmGmCmAmAmGUUmAAmAmAUmAmAmGmGCUmAGUCmCGUUmAmUm
CmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCsmU
smUsmU;
(SEQ ID NOs: 572)
mAsmUsmCsGACAAGAAAGGGACUGAmGUUUUAGmAmGmCmUmAmGmAmAmAmUmAmGmCmA
mAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmGmAmAmAmAmAmG
mUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
(SEQ ID NOs: 573)
mCsmAsmUsCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsU;
(SEQ ID NOs: 574)
mAsmUsmCsmGmAmCmAmAmGmAfAfAfGIGGsAsfCUGmAmGUSUUfUfAmGmAmGmCmUm
AmGmAmAmAmUmAmGmCmAmAmGUUmAAmAmAUmAmAmGmGCUmAGUCmCGUUmAmUmCmAm
AmCmUmUmGmAmAmAmAmAmGUGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCsmUsmUsmU;
(SEQ ID NOs: 575)
mAsmUsmCsGACAAGAAAGGGACUGAmGUUUUAGmAmGmCmUmAmGmAmAmAmUmAmGmCmA
mAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmGmAmAmAmAmAmG
UGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
(SEQ ID NOs: 576)
mAsmUsmCsmGmAmCmAmAmGmAAAGGGACUGmAmGUUUUAmGmAmGmCmUmAmGmAmAmAm
UmAmGmCmAmAmGUUmAAmAmAUmAmAmGmGCUmAGUCmCGUUmAmUmCmAmAmCmUmUmGm
AmAmAmAmAmGUGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCsmUsmUsmU;
(SEQ ID NOs: 577)
mAsmUsmCsmGmAmCmAmAmGmAAAGGGACUGAmGUUUUAGmAmGmCmUmAmGmAmAmAmUm
AmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmGmAmAm
AmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
(SEQ ID NOs: 578)
mCsmAsmUsmCmGmAmCmAmAmGmAAAGGGACUGAmGUUUUAGmAmGmCmUmAmGmAmAmAm
UmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmGmAm
AmAmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
(SEQ ID NOs: 579)
mAsmUsmCsmGmAmCmAmAmGmAmAAmGmGGAmCUGmAmGUUUUAGmAmGmCmUmAmGmAmA
mAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmG
mAmAmAmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
(SEQ ID NOs: 580)
mCsmAsmUsmCmGmAmCmAmAmGmAmAAmGmGGAmCUGmAmGUUUUAGmAmGmCmUmAmGmA
mAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmU
mGmAmAmAmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
(SEQ ID NOs: 581)
mCsmAsmUsCGACAAGAAAGGGACUGAmGUUUUAGmAmGmCmUmAmGmAmAmAmUmAmGmCm
AmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmGmAmAmAmAmAm
GUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
and
(SEQ ID NOs: 582)
mAsmUsmCsGACAAGAAAGGGACUGAGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmC
mCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmG
mGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
(SEQ ID NOs: 583)
mAsmUsmCsmGmAmCmAmAmGmAAAGGGACUGAGUUUUAGAmGmCmCmGmGmCmGmGmAmAm
AmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAm
AmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
(SEQ ID NOs: 584)
mAsmUsmCsGACAAGAAAGGGACUGAGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGU
UAAAAUAAGGCUAGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAmGmUmGGmCmAmC
mCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU;
or
(SEQ ID NO: 585)
mAsmUsmCsGACAAGAAAGGGACUGAGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmC
mCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAm
GmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU;
wherein “N” represents any nucleotide, “mN” indicates a 2′-OMe modification of the nucleotide “N”, fN indicates a 2′-fluoro (F) modification of the nucleotide “N,” and “Ns” indicates that the nucleotide “N” is linked to the following nucleotide by a phosphorothioate. sgRNA modified sequence here.
22 . A pharmaceutical composition comprising the base editor system of claim 19 and a pharmaceutically acceptable carrier, vehicle, or excipient.
23 . The pharmaceutical composition of claim 22 further comprising a lipid.
24 . The pharmaceutical composition of claim 23 , wherein the lipid is a cationic lipid.
25 . The pharmaceutical composition of claim 22 further comprising a lipid nanoparticle.
26 . The pharmaceutical composition of claim 25 , wherein the nucleic acid encoding the base editor is an mRNA molecule, and wherein the mRNA molecule and the sgRNA are formulated in the lipid nanoparticle.
27 . A method of editing an alpha-1 antitrypsin polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency, the method comprising contacting the polynucleotide with a guide RNA, and a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and an adenosine deaminase domain, wherein the napDNAbp domain comprises an alteration selected from the group consisting of M1135L, E1250K, A1283D, Q1136Y, R1337K, R765A, and Q768A of an amino acid sequence, or a fragment thereof lacking an N-terminal methionine, that is at least 90% identical to:
(SEQ ID NO: 554)
MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT
RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD
EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI
QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL
TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT
EITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF
YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLK
DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT
NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK
VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV
LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDF
LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV
DELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL
QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD
NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV
VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN
GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNS
DKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP
IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKGNELALPSKYVNFLYLAS
HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI
REQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQ
LGGD,
and wherein said guide RNA targets said base editor to effect an alteration of the SNP associated with alpha-1 antitrypsin deficiency.
28 . A method of editing an alpha-1 antitrypsin polynucleotide comprising a single nucleotide polymorphism (SNP) associated with alpha-1 antitrypsin deficiency in a cell, the method comprising contacting the cell with the base editor system of claim 19 .
29 . A method for treating alpha-1 antitrypsin deficiency (A1AD) in a subject, the method comprising: administering to the subject the pharmaceutical composition of claim 22 to effect an A·T to G·C alteration of a single nucleotide polymorphism (SNP) associated with A1AD, thereby treating A1AD in the subject.
30 . The method of claim 29 , wherein the SNP associated with A1AD results in expression of an A1AT polypeptide comprising an amino acid alteration selected from the group consisting of E264V and E342K.