COMPOSITIONS AND METHODS FOR ENGRAFTMENT OF BASE EDITED CELLS
The invention provides compositions comprising novel adenosine base editors (e.g., ABE8) that have increased efficiency and methods of using these adenosine deaminase variants for editing a target sequence and methods of using same to treat genetical disorder or conditions, e.g. sickle cell disease, with engraftment.
1 . A method of engrafting nucleobase-edited hematopoietic stem cells or progenitors thereof in a subject having a hemoglobinopathy, the method comprising:
(a) contacting hematopoietic stem cells or progenitors thereof in vitro with a guide RNA and a base editor comprising a polynucleotide programmable DNA binding domain and a deaminase domain, or a polynucleotide encoding the base editor, wherein the guide RNA targets the polynucleotide programmable DNA binding domain to induce a nucleobase change in a target hemoglobin (HBB) gene or in the promoter region of HBG1/2, thereby obtaining nucleobase-edited hematopoietic stem cells or progenitors thereof; and wherein the nucleobase-edited hematopoietic stem cells or progenitors thereof are contacted with the gRNA and the base editor within 48 hours following collection from a donor; and
(b) administering the nucleobase-edited hematopoietic stem cells or progenitors thereof to a subject in an effective amount to obtain engraftment of the nucleobase-edited hematopoietic stem cells or progenitors thereof in tissues of the subject after administration.
2 . The method of claim 1 , wherein the nucleobase-edited hematopoietic stem cells or progenitors thereof comprise CD34 + cells enriched from polymorphonuclear blood cells (PBMCs) collected from the donor.
3 . A method of engrafting nucleobase-edited hematopoietic stem cells or progenitors thereof in a subject having a hemoglobinopathy or treating a hemoglobinopathy in a subject, the method comprising:
(a) contacting hematopoietic stem cells or progenitors thereof in vitro with a guide RNA and a base editor comprising a polynucleotide programmable DNA binding domain and a deaminase domain, or a polynucleotide encoding the base editor, wherein the guide RNA targets the polynucleotide programmable DNA binding domain to induce a nucleobase change in a target hemoglobin (HBB) gene or in the promoter region of HBG1/2, thereby obtaining nucleobase-edited hematopoietic stem cells or progenitors thereof; and
(b) administering the nucleobase-edited hematopoietic stem cells or progenitors thereof to a subject in an effective amount to obtain engraftment of the nucleobase-edited hematopoietic stem cells or progenitors thereof in tissues of the subject after administration.
4 . (canceled)
5 . The method of claim 1 , wherein the nucleobase change is an A to G nucleobase change.
6 . The method of claim 1 , wherein the deaminase domain is an adenosine deaminase domain and shares at least 85% sequence identity with the sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG MNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 3), and wherein the adenosine deaminase domain is capable of catalyzing the hydrolytic deamination of adenine or adenosine.
7 - 8 . (canceled)
9 . The method of claim 6 , wherein the adenosine deaminase domain comprises a combination of alterations selected from the group consisting of:
Y147R, Q154R, and Y123H;
Y147R, Q154R, and I76Y;
Y147R, Q154R, and T166R;
Y147T and Q154R; Y147T and Q154S; and
Y123H, Y147R, Q154R, and I76Y.
10 . (canceled)
11 . The method of claim 1 , wherein the deaminase domain is a TadA*8 variant.
12 . The method of claim 11 , wherein the TadA*8 variant is selected from the group consisting of: TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, and TadA*8.13; and.
wherein the base editor is an ABE8 base editor selected from the group consisting of: ABE8.1, ABE8.2, ABE8.3, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE13.
13 . (canceled)
14 . A method of engrafting nucleobase-edited hematopoietic stem cells or progenitors thereof in a subject having a hemoglobinopathy or treating a hemoglobinopathy in a subject, the method comprising:
(a) contacting hematopoietic stem cells or progenitors thereof in vitro with a guide RNA and an adenosine base editor comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain comprising an amino acid sequence with at least 85% sequence identity to the sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG MNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 3) and comprising the alterations Y123H, Y147R, and Q154R, or a polynucleotide encoding the base editor, wherein the adenosine deaminase domain catalyzes the hydrolytic deamination of adenine or adenosine, and wherein said guide RNA targets said polynucleotide programmable DNA binding domain to induce an A to G nucleobase change in a target hemoglobin (HBB) gene or in the promoter region of HBG1/2, thereby obtaining nucleobase-edited hematopoietic stem cells or progenitors thereof; and
(b) administering the nucleobase-edited hematopoietic stem cells or progenitors thereof to a subject in an effective amount to obtain engraftment of the nucleobase-edited hematopoietic stem cells or progenitors thereof in tissues of the subject after administration.
15 . (canceled)
16 . The method of claim 6 , wherein the adenosine deaminase domain comprises an alteration at position 82 or 166.
17 . The method of claim 16 , wherein the alteration at position 82 is V82S and the alteration at position 166 is T166R.
18 - 21 . (canceled)
22 . The method of claim 1 , wherein the polynucleotide programmable DNA binding domain is a Cas9.
23 . The method of claim 22 , wherein the Cas9 is a SpCas9, a SaCas9, or a variant thereof.
24 - 26 . (canceled)
27 . The method of claim 1 , wherein the polynucleotide programmable DNA binding domain is a nickase.
28 - 31 . (canceled)
32 . A method of engrafting edited hematopoietic stem cells or progenitors thereof in a subject having a hemoglobinopathy or treating a subject having a hemoglobinopathy, the method comprising:
(a) contacting hematopoietic stem cells or progenitors thereof in vitro with a guide RNA and a base editor comprising an amino acid sequence with at least 80% sequence identity to one of the following two amino acid sequences: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPES SGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYE YFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKA QVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTITQKGQK NSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVD HIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKA ERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGDEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 258), and MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPE SSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLM DVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGT SESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK PILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TIQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQR KFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTITIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 259), or a polynucleotide encoding the base editor, wherein said guide RNA targets said polynucleotide programmable DNA binding domain to induce an A to G nucleobase change in the promoter region of HBG1/2, thereby obtaining edited hematopoietic stem cells or progenitors thereof;
(b) administering the nucleobase-edited hematopoietic stem cells or progenitors thereof to a subject in an effective amount to obtain engraftment of the nucleobase-edited hematopoietic stem cells or progenitors thereof in tissues of the subject after administration.
33 - 46 . (canceled)
47 . The method of claim 1 , wherein the hematopoietic stem cells or progenitors thereof comprise a single-nucleotide polymorphism (SNP) associated with sickle cell disease (SCD).
48 . The method of claim 47 , wherein the SNP associated with SCD results in a E6V substitution in a hemoglobin beta unit encoded by the HBB gene.
49 - 54 . (canceled)
55 . The method of claim 1 , wherein the nucleobase change results in a E6A substitution in the hemoglobin beta unit encoded by the HBB gene.
56 - 64 . (canceled)
65 . The method of claim 1 , wherein levels of fetal hemoglobin (HbF) are increased in the subject following engraftment relative to the levels in a control subject that received unedited hematopoietic stem cells or progenitors thereof.
66 - 70 . (canceled)
71 . The method of claim 1 , wherein the nucleobase-edited hematopoietic stem cells or progenitors thereof express HbF.
72 - 75 . (canceled)
76 . The method of claim 1 , wherein the subject has sickle cell disease (SCD), thalassemia, and/or anemia.
77 - 80 . (canceled)
81 . The method of claim 1 , wherein the nucleobase change abolishes, disrupts, or reduces BCL11A binding in the promoter region of HBG1/2.
82 . (canceled)
83 . The method of claim 1 , wherein the nucleobase change is associated with an increase in expression of HBG1/2.
84 - 94 . (canceled)
95 . The method of claim 1 , wherein the gRNA comprises or consists of the sequence, from 5′-3′:
GACCAAUAGCCUUGACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC
UAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU,
corresponding to bases 4-97 of SEQ ID NO: 129;
(SEQ ID NO: 129)
from 5′-3′: csususGACCAAUAGCCUUGACAGUUUUAGAGCUAGAAAU
AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG
UCGGUGCUsususu ,
wherein lowercase characters indicate 2′-O-methylated
nucleobases, and “s” indicates phosphorothioates
(SEQ ID NO: 129);
or
(SEO ID NO: 126)
gsascsUUCUCCACAGGAGUCAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAA
AUAAGGCUAGUCCGUUAUUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsu
susu -3′,
(SEO ID NO: 127)
5′- ascsusUCUCCACAGGAGUCAGGGUUUUAGAGCUAGAAAUAGCAAGUUA
AAAUAAGGCUAGUCCGUUAUUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU
sususu -3′,
and
(SEQ ID NO: 128)
5′-
csususCUCCACAGGAGUCAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU
AAGGCUAGUCCGUUAUUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsusu
su -3′,
wherein lowercase characters indicate 2′-O-methvlated
nucleobases, and “s” indicates phosphorothioates.
96 - 113 . (canceled)
114 . A kit for use in the method of claim 1 , wherein the kit comprises the guide RNA and a polynucleotide encoding the base editor.