COMPOSITIONS AND METHODS FOR TREATING HEPATITIS B VIRUS INFECTION
The invention features compositions and methods for introducing mutations into the hepatitis B virus (HBV) genome.
1 . A method of editing a nucleobase of a hepatitis B virus (HBV) genome, the method comprising contacting the HBV genome with one or more guide RNAs and a base editor comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase, wherein said guide RNA targets said base editor to effect an alteration of the nucleobase of the HBV genome, and wherein said one or more guide RNAs comprise a nucleotide sequence, from 5′ to 3′, or a 1, 2, 3, 4, or 5 nucleotide 5′ and/or 3′ truncation fragment thereof, selected from one or more of ACAAGAAUCCUCACAAUACC (SEQ ID NO:
(SEQ ID NO: 405)
ACAAGAAUCCUCACAAUACC,
(SEQ ID NO: 406)
UCGCUGGAUGUGUCUGCGGC,
(SEQ ID NO: 407)
CACCUGUAUUCCCAUCCCAU,
(SEQ ID NO: 408)
GGGGCCAAGUCUGUACAGCA,
(SEQ ID NO: 409)
GCUGACGCAACCCCCACUGG,
(SEQ ID NO: 410)
GGAGCUACUGUGGAGUUACU,
(SEQ ID NO: 411)
UUCUUCUAGGGGACCUGCCU,
(SEQ ID NO: 412)
GAGGACAAACGGGCAACAUA,
(SEQ ID NO: 413)
UUGUCAACAAGAAAAACCCC,
(SEQ ID NO: 414)
CCCAAGGUCUUACAUAAGAG,
(SEQ ID NO: 415)
CCGGGCAACGGGGUAAAGGU,
(SEQ ID NO: 416)
ACACAGAAAGGCCUUGUAAG,
(SEQ ID NO: 10834)
GAAAGCCCAAGAUGAUGGGA,
and
(SEQ ID NO: 417)
CACGCACGCGCUGAUGGCCC.
2 . A method of editing a nucleobase of a hepatitis B virus (HBV) genome, the method comprising:
(i) contacting a cell comprising an HBV genome with one or more guide RNAs and a base editor comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase or cytidine deaminase, wherein said guide RNA targets said base editor to effect an alteration of the nucleobase of the HBV genome, thereby editing a nucleobase in the HBV genome; and
(ii) contacting the cell with an antiretroviral drug, wherein contacting the cell with the antiretroviral drug is associated with an increase in base editing efficiency relative to a reference cell not contacted with the antiretroviral drug.
3 . A method reducing HBsAg produced from integrated HBV DNA, the method comprising contacting a cell comprising an HBV genome with one or more guide RNAs and a base editor comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase or cytidine deaminase, wherein said guide RNA targets said base editor to effect an alteration of the nucleobase of the HBV genome, thereby reducing HBsAg production from an integrated HBV DNA.
4 . The method of claim 2 , wherein the guide RNA is selected from one or more of
(SEQ ID NO: 405)
ACAAGAAUCCUCACAAUACC,
(SEQ ID NO: 406)
UCGCUGGAUGUGUCUGCGGC,
(SEQ ID NO: 407)
CACCUGUAUUCCCAUCCCAU,
(SEQ ID NO: 408)
GGGGCCAAGUCUGUACAGCA,
(SEQ ID NO: 409)
GCUGACGCAACCCCCACUGG,
(SEQ ID NO: 410)
GGAGCUACUGUGGAGUUACU,
(SEQ ID NO: 411)
UUCUUCUAGGGGACCUGCCU,
(SEQ ID NO: 412)
GAGGACAAACGGGCAACAUA,
(SEQ ID NO: 413)
UUGUCAACAAGAAAAACCCC,
(SEQ ID NO: 414)
CCCAAGGUCUUACAUAAGAG,
(SEQ ID NO: 415)
CCGGGCAACGGGGUAAAGGU,
(SEQ ID NO: 416)
ACACAGAAAGGCCUUGUAAG,
and
(SEQ ID NO: 417)
CACGCACGCGCUGAUGGCCC;
wherein the one or more guide RNAs comprise a spacer sequence selected from a sequence listed in Table 2A, Table 2B, Table 2C, and/or SEQ ID NOs: 3105-5485 and 8220-10830; or
wherein the one or more guide RNAs comprise the nucleotide sequences
(SEQ ID NO: 424)
mG*mA*mA*AGCCCAGGAUGAUGGGAGUUUUAGAGCUAGAAAUAGCAAG
UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG
GUGCUmU*mU*mU*
and
(SEQ ID NO: 422)
mC*mC*mA*UGCCCCAAAGCCACCCAGUUUUAGAGCUAGAAAUAGCAAG
UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG
GUGCUmU*mU*mU*.
5 . The method of claim 2 , wherein the one or more guide RNAs comprise the following nucleotide sequences gsasasAGCCCAGGAUGAUGGGAgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGU ccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 424) and cscsasUGCCCCAAAGCCACCCAgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGU CcGUUAucAAcuugaaaaagugGcaccgagucggugcusususu (SEQ ID NO: 422), where a, c, u, or g represent an A, C, U, or G nucleotide containing a 2′O-methyl (M) modification, respectively; and as, cs, us, or gs represent an A, C, U, or G nucleotide containing a 2′-O-methyl 3′-phosphorothioate (MS) modification, respectively.
6 . A method of editing a nucleobase of a hepatitis B virus (HBV) genome, the method comprising contacting a cell comprising an HBV genome with an mRNA encoding a base editor comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase or cytidine deaminase, and one or more guide RNAs comprising the sequences GAAAGCCCAGGAUGAUGGGA (SEQ ID NO: 657) and CCAUGCCCCAAAGCCACCCA (SEQ ID NO: 662), wherein said one or more guide RNAs target said base editor to effect an alteration of the nucleobase of the HBV genome, thereby editing a nucleobase in the HBV genome.
7 . The method of claim 2 , wherein alteration of the nucleobase in the polynucleotide encoding the HBV protein results in a missense mutation.
8 . The method of claim 7 , wherein the missense mutation is in an HBV pol gene and wherein the missense mutation results in an E24G, L25F, P26F, R27C, V48A, V48I, S382F, V378I, V378A, V379I, V379A, L377F, D380G, D380N, F381P, R376G, A422T, F423P, A432V, M433V, P434S, D540G, A688V, D689G, A717T, E718K, P713S, P713L, or L719P in an HBV polymerase protein encoded by the HBV pol gene:
wherein the missense mutation is in an HBV core gene, and wherein the missense mutation results in a T160A, T160A, P161F, S162L, C183R, or *184Q in an HBV core protein encoded by the HBV core gene;
wherein the missense mutation is in an HBV X gene, and wherein the missense mutation results in a H86R, W120R, E122K, E121K, or L141P in an HBV X protein encoded by the HBV X gene; and/or
wherein the missense mutation is in an HBV S gene, and wherein the missense mutation results in a S38F, L39F, W35R, W36R, T37I, T37A, R78Q, S34L, F80P, or D33G in an HBV S protein encoded by the HBV S gene.
9 . A method of treating hepatitis B virus (HBV) infection in a subject comprising administering to a subject in need thereof a fusion protein, protein complex, or polynucleotide encoding said fusion protein or protein complex, said fusion protein or protein complex comprising a polynucleotide programmable DNA binding domain and a base editor domain that is an adenosine deaminase domain, and one or more guide RNAs that target the base editor domain to effect an A·T to G·C alteration of a nucleobase of the HBV genome, wherein said one or more guide RNAs comprise a nucleotide sequence, from 5′ to 3′, or a 1, 2, 3, 4, or 5 nucleotide 5′ and/or 3′ truncation fragment thereof, selected from one or more of
(SEQ ID NO: 405)
ACAAGAAUCCUCACAAUACC,
(SEQ ID NO: 406)
UCGCUGGAUGUGUCUGCGGC,
(SEQ ID NO: 407)
CACCUGUAUUCCCAUCCCAU,
(SEQ ID NO: 408)
GGGGCCAAGUCUGUACAGCA,
(SEQ ID NO: 409)
GCUGACGCAACCCCCACUGG,
(SEQ ID NO: 410)
GGAGCUACUGUGGAGUUACU,
(SEQ ID NO: 411)
UUCUUCUAGGGGACCUGCCU,
(SEQ ID NO: 412)
GAGGACAAACGGGCAACAUA,
(SEQ ID NO: 413)
UUGUCAACAAGAAAAACCCC,
(SEQ ID NO: 414)
CCCAAGGUCUUACAUAAGAG,
(SEQ ID NO: 415)
CCGGGCAACGGGGUAAAGGU,
(SEQ ID NO: 416)
ACACAGAAAGGCCUUGUAAG,
and
(SEQ ID NO: 417)
CACGCACGCGCUGAUGGCCC.
10 . A method of treating a hepatitis B virus (HBV) infection in a subject, comprising administering to a subject in need thereof with one or more guide RNAs and a base editor comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase or cytidine deaminase, wherein said one or more guide RNAs comprise the sequences GAAAGCCCAGGAUGAUGGGA (SEQ ID NO: 657) and CCAUGCCCCAAAGCCACCCA (SEQ ID NO: 662) that target said base editor to effect an alteration of the nucleobase of the HBV genome, thereby editing a nucleobase in the HBV genome.
11 . A composition comprising a base editor(s) bound to a guide RNA(s), wherein said guide RNA(s) comprises a nucleotide sequence, from 5′ to 3′, or a 1, 2, 3, 4, or 5 nucleotide 5′ and/or 3′ truncation fragment thereof, selected from one or more of
(SEQ ID NO: 405)
ACAAGAAUCCUCACAAUACC,
(SEQ ID NO: 406)
UCGCUGGAUGUGUCUGCGGC,
(SEQ ID NO: 407)
CACCUGUAUUCCCAUCCCAU,
(SEQ ID NO: 408)
GGGGCCAAGUCUGUACAGCA,
(SEQ ID NO: 409)
GCUGACGCAACCCCCACUGG,
(SEQ ID NO: 410)
GGAGCUACUGUGGAGUUACU,
(SEQ ID NO: 411)
UUCUUCUAGGGGACCUGCCU,
(SEQ ID NO: 412)
GAGGACAAACGGGCAACAUA,
(SEQ ID NO: 413)
UUGUCAACAAGAAAAACCCC,
(SEQ ID NO: 414)
CCCAAGGUCUUACAUAAGAG,
(SEQ ID NO: 415)
CCGGGCAACGGGGUAAAGGU,
(SEQ ID NO: 416)
ACACAGAAAGGCCUUGUAAG,
and
(SEQ ID NO: 417)
CACGCACGCGCUGAUGGCCC.
12 . The composition of claim 11 , wherein the guide RNAs comprise the following scaffold sequence:
gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugG caccgagucggugcusususu (SEQ ID NO: 317), wherein a, c, u, or g represents bases with a 2′O-methyl (M) modification, and as, cs, us, or gs represents bases with a 2′-O-methyl 3′-phosphorothioate (MS) modification.
13 . The composition of claim 11 , wherein the base editor comprises an adenosine deaminase.
14 . A pharmaceutical composition for the treatment of HBV infection comprising:
(i) a base editor, or a nucleic acid encoding the base editor, and one or more guide RNAs (gRNA) comprising a nucleic acid sequence complementary to an HBV gene in a pharmaceutically acceptable excipient, wherein said one or more guide RNAs comprise a nucleotide sequence, from 5′ to 3′, or a 1, 2, 3, 4, or 5 nucleotide 5′ and/or 3′ truncation fragment thereof, selected from one or more of ACAAGAAUCCUCACAAUACC (SEQ ID NO: 405), UCGCUGGAUGUGUCUGCGGC (SEQ ID NO:406), CACCUGUAUUCCCAUCCCAU (SEQ ID NO: 407), GGGGCCAAGUCUGUACAGCA (SEQ ID NO: 408), GCUGACGCAACCCCCACUGG (SEQ ID NO: 409), GGAGCUACUGUGGAGUUACU (SEQ ID NO: 410), UUCUUCUAGGGGACCUGCCU (SEQ ID NO: 411), GAGGACAAACGGGCAACAUA (SEQ ID NO: 412), UUGUCAACAAGAAAAACCCC (SEQ ID NO: 413), CCCAAGGUCUUACAUAAGAG (SEQ ID NO: 414), CCGGGCAACGGGGUAAAGGU (SEQ ID NO: 415), ACACAGAAAGGCCUUGUAAG (SEQ ID NO: 416), and CACGCACGCGCUGAUGGCCC (SEQ ID NO: 417).
15 . The pharmaceutical composition of claim 14 , wherein the base editor
(i) comprises a nuclease inactive bhCas12b; or
(ii) comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
(SEQ ID NO: 418)
MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA
LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHP
GMNHRVEITEGILADECAALLCRFFRMPRRVENAQKKAQSSTDGSSGSETPGTSESATPESS
GAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAI
YEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVE
KKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDP
LAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWES
WNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLR
GWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPY
LYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKL
TVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGT
LGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKP
KELTEWIKDSKGKKLKSGIESLEIGLRVMSIALGQRQAAAASIFEVVDQKPDIEGKLFFPIK
GTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITERE
KRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKS
LSDGRKGLYGISLKNIDEIDRTRKELLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKED
RLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYKERSRFENSRLMKWSR
REIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCRVVTKEKLQDNRFFKNLQREGR
LTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWIRTHGFYKVYCK
AYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDS
DILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIE
DDSSKQSMKRPAATKKAGQAKKKK.
16 . The pharmaceutical composition of claim 14 , wherein the base editor comprises a bhCas12b, or a bhCas12b variant comprising amino acid substitutions D952A, S893R, K846R, and E837G.
17 . The composition of claim 14 , wherein the guide RNAs comprise the following scaffold sequence:
gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugG caccgagucggugcusususu (SEQ ID NO: 317), wherein a, c, u, or g represents bases with a 2′O-methyl (M) modification, and as, cs, us, or gs represents bases with a 2′-O-methyl 3′-phosphorothioate (MS) modification.
18 . The composition of claim 14 , wherein the guide RNAs comprise the following scaffold sequence:
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGCUmU*mU*mU* (SEQ ID NO: 422), where mA*, mC*, mG*, and mU* represent bases with a 2′-O-methyl 3′-phosphorothioate modification.
19 . A method of treating HBV infection, the method comprising administering to a subject in need thereof the composition of claim 14 .
20 . A guide RNA comprise a nucleotide sequence, from 5′ to 3′, or a 1, 2, 3, 4, or 5 nucleotide 5′ and/or 3′ truncation fragment thereof, selected from one or more of
(SEQ ID NO: 405)
ACAAGAAUCCUCACAAUACC,
(SEQ ID NO: 406)
UCGCUGGAUGUGUCUGCGGC,
(SEQ ID NO: 407)
CACCUGUAUUCCCAUCCCAU,
(SEQ ID NO: 408)
GGGGCCAAGUCUGUACAGCA,
(SEQ ID NO: 409)
GCUGACGCAACCCCCACUGG,
(SEQ ID NO: 410)
GGAGCUACUGUGGAGUUACU,
(SEQ ID NO: 411)
UUCUUCUAGGGGACCUGCCU,
(SEQ ID NO: 412)
GAGGACAAACGGGCAACAUA,
(SEQ ID NO: 413)
UUGUCAACAAGAAAAACCCC,
(SEQ ID NO: 414)
CCCAAGGUCUUACAUAAGAG,
(SEQ ID NO: 415)
CCGGGCAACGGGGUAAAGGU,
(SEQ ID NO: 416)
ACACAGAAAGGCCUUGUAAG,
and
(SEQ ID NO: 417)
CACGCACGCGCUGAUGGCCC.
21 . The guide RNA of claim 20 , wherein the guide RNA comprises the following scaffold sequence:
gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugG caccgagucggugcusususu (SEQ ID NO: 317), wherein a, c, u, or g represents bases with a 2′O-methyl (M) modification, and as, cs, us, or gs represents bases with a 2′-O-methyl 3′-phosphorothioate (MS) modification.
22 . The guide RNA of claim 21 , wherein the guide RNA comprises the following scaffold sequence:
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGCUmU*mU*mU* (SEQ ID NO: 422), where mA*, mC*, mG*, and mU* represent bases with a 2′-O-methyl 3′-phosphorothioate modification.
23 . A pharmaceutical composition comprising (i) a nucleic acid encoding a base editor, and (ii) the guide RNA of claim 20 .