IP Library › Patent Application 18507980

Patent Application

App. No. 18/507,980

COMPOSITIONS AND METHODS FOR TREATING TRANSTHYRETIN AMYLOIDOSIS

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Patent No.: US None
App. No.: 18/507,980

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Abstract

The invention features compositions and methods for editing a transthyretin polynucleotide sequence to treat amyloidosis.

Claims (530)

1 . A method for editing a transthyretin (TTR) polynucleotide sequence, the method comprising: contacting the polynucleotide sequence with a guide RNA and a base editor comprising a polynucleotide programmable DNA binding polypeptide and an adenosine or cytidine deaminase, wherein said guide RNA targets said base editor to effect an alteration of a nucleobase of the TTR polynucleotide sequence.

2 . The method of claim 1 , wherein the editing introduces an alteration that corrects a mutation in a TTR polynucleotide and/or wherein the editing introduces an alteration that reduces or eliminates expression of a TTR polypeptide.

3 . The method of claim 1 , wherein the alteration is in a splice acceptor, splice donor, intronic sequence, exonic sequence, enhancer, or promoter.

4 . A method for editing a transthyretin (TTR) polynucleotide sequence, the method comprising: contacting the polynucleotide sequence with a guide RNA and a fusion protein comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an arginine (R) or a threonine (T) at amino acid position 147 of the following amino acid sequence, and the adenosine deaminase domain has at least about 85% sequence identity to the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 4; TadA*7.10), or wherein the cytidine deaminase domain comprises an amino acid sequence with at least about 85% sequence identity to the amino acid sequence: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEV NFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNR QGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPC LNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 15; BE4 cytidine deaminase domain), wherein said guide RNA targets said fusion protein to effect an alteration of a nucleobase of the TTR polynucleotide sequence.

5 . The method of claim 4 , wherein the alteration is in a region of the TTR promoter corresponding to nucleotide positions +1 to −225 of the TTR promoter, wherein position +1 corresponds to A of the start codon (ATG) of the TTR polynucleotide sequence;

wherein the alteration is in a region of the TTR promoter corresponding to nucleotide positions +1 to −198 of the TTR promoter, wherein position +1 corresponds to A of the start codon (ATG) of the TTR polynucleotide sequence; wherein the alteration is in a region of the TTR promoter corresponding to nucleotide positions +1 to −177 of the TTR promoter, wherein position +1 corresponds to A of the start codon (ATG) of the TTR polynucleotide sequence; or

wherein the alteration is in a region of the TTR promoter corresponding to nucleotide positions −106 to −176 of the TTR promoter, wherein position +1 corresponds to A of the start codon (ATG) of the TTR polynucleotide sequence.

6 . The method of claim 1 , wherein the TTR polynucleotide sequence encodes a mature TTR polypeptide comprising a pathogenic alteration selected from the group consisting of T60A, V30M, V30A, V30G, V30L, V122I, and V122A.

7 . The method of claim 1 , wherein the altered nucleobase is

4A of the nucleotide sequence TATAGGAAAACCAGTGAGTC (SEQ ID NO: 425; TSBT×2602/gRNA1598 target site sequence corresponding to sgRNA_361);

6A of the nucleotide sequence TACTCACCTCTGCATGCTCA (SEQ ID NO: 426; TSBT×2603/gRNA1599 target site sequence corresponding to sgRNA_362);

5A of the nucleotide sequence ACTCACCTCTGCATGCTCAT (SEQ ID NO: 427; TSBT×2604/gRNA1606 target site sequence corresponding to sgRNA_363);

7A of the nucleotide sequence ATACTCACCTCTGCATGCTCA (SEQ ID NO: 429; TSBT×2606 target site sequence corresponding to sgRNA_365);

6A of the nucleotide sequence TTGGCAGGATGGCTTCTCATCG (SEQ ID NO: 431; TSBT×2608/gRNA-#19 target site corresponding to sgRNA_367);

9A of the sequence TTGGCAGGATGGCTTCTCATCG (SEQ ID NO: 431; TSBT×2608/gRNA-#19 target site corresponding to sgRNA_367);

5A of the sequence GGCTATCGTCACCAATCCCA (SEQ ID NO: 439; corresponding to sgRNA_375); or

4A of the sequence GCTATCGTCACCAATCCCAA (SEQ ID NO: 440; corresponding to sgRNA_376);

7C of the nucleotide sequence TACTCACCTCTGCATGCTCA (SEQ ID NO: 426; TSBT×2603/gRNA1599 target site corresponding to sgRNA_362);

6C of the nucleotide sequence ACTCACCTCTGCATGCTCAT (SEQ ID NO: 427; TSBT×2604/gRNA1606 target site corresponding to sgRNA_363);

7C of the nucleotide sequence TACCACCTATGAGAGAAGAC (SEQ ID NO: 428; TSBT×2605 target site corresponding to sgRNA_364);

8C of the nucleotide sequence ATACTCACCTCTGCATGCTCA (SEQ ID NO: 429; TSBT×2606 target site corresponding to sgRNA 365); or

11C of the nucleotide sequence ACTGGTTTTCCTATAAGGTGT (SEQ ID NO: 430; TSBT×2607 target site corresponding to sgRNA_366).

8 . The method of claim 4 , wherein the TadA deaminase is TadA*7.10, TadA*8.1, TadA*8.2, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.15, TadA*8.16, TadA*8.19, TadA*8.20, TadA*8.21, or TadA*8.24.

9 . The method of claim 1 , wherein the guide RNA(s) comprises a nucleotide sequence selected from one or more of those sequences listed in Table 1, Table 2A, or Table 2B; or any of the aforementioned sequences wherein 1, 2, 3, 4, or 5 nucleotides is deleted from the 5′ and/or 3′ terminus of the nucleotide sequence.

10 . The method of claim 1 , wherein the guide RNA(s) comprises a nucleotide sequence, selected from the group consisting of:

(SEQ ID NO: 408; sgRNA_361/gRNA1598)

5′-UAUAGGAAAACCAGUGAGUC-3′;

(SEQ ID NO: 409; sgRNA_362/gRNA1599)

5′-UACUCACCUCUGCAUGCUCA-3′;

(SEQ ID NO: 410; sgRNA_363/gRNA1606)

5′-ACUCACCUCUGCAUGCUCAU-3′;

(SEQ ID NO: 412; sgRNA_365)

5′-AUACUCACCUCUGCAUGCUCA-3′;

(SEQ ID NO: 414; sgRNA_367/gRNA-#19)

5′-UUGGCAGGAUGGCUUCUCAUCG-3′;

(SEQ ID NO: 422; sgRNA_375)

5′-GGCUAUCGUCACCAAUCCCA-3′;

(SEQ ID NO: 423; sgRNA_376)

5′-GCUAUCGUCACCAAUCCCAA-3′;

(SEQ ID NO: 561; gRNA1604)

5′-ACACCUUAUAGGAAAACCAG-3′;

(SEQ ID NO: 554; gRNA1597)

5′-CUCUCAUAGGUGGUAUUCAC-3′;

(SEQ ID NO: 557; gRNA1600)

5′-GCAACUUACCCAGAGGCAAA-3′;

(SEQ ID NO: 551; gRNA1594)

5′-CAACUUACCCAGAGGCAAAU-3′;

(SEQ ID NO: 558; gRNA1601)

5′-UCUGUAUACUCACCUCUGCA-3′;

(SEQ ID NO: 462; gRNA1756)

5′-CAAAUAUGAACCUUGUCUAG-3′;

(SEQ ID NO: 470; gRNA1764)

5′-GAACCUUGUCUAGAGAGAUU-3′;

(SEQ ID NO: 492; gRNA1786)

5′-UGAGUAUAAAAGCCCCAGGC-3′;

and

(SEQ ID NO: 478; gRNA1772)

5′-GCCAUCCUGCCAAGAAUGAG-3′;

(SEQ ID NO: 409; sgRNA_362/gRNA1599)

5′-UACUCACCUCUGCAUGCUCA-3′,

(SEQ ID NO: 410; sgRNA_363/gRNA1606)

5′-ACUCACCUCUGCAUGCUCAU-3′,

(SEQ ID NO: 411; sgRNA_364)

5′-UACCACCUAUGAGAGAAGAC-3′,

(SEQ ID NO: 412; sgRNA_365)

5′-AUACUCACCUCUGCAUGCUCA-3′,

(SEQ ID NO: 413; sgRNA_366)

5′-ACUGGUUUUCCUAUAAGGUGU-3′,

(SEQ ID NO: 551; gRNA1594)

5′-CAACUUACCCAGAGGCAAAU-3′,

and

(SEQ ID NO: 496; gRNA1790)

5′-UGUUGACUAAGUCAAUAAUC-3′;

or any of the aforementioned sequences wherein 1, 2, 3, 4, or 5 nucleotides is deleted from the 5′ and/or 3′ terminus of the nucleotide sequence.

11 . A method for editing a transthyretin (TTR) polynucleotide sequence, the method comprising: contacting the polynucleotide sequence with a guide RNA and a Cas12b endonuclease, wherein said guide RNA targets said endonuclease to effect a double-stranded break of the TTR polynucleotide sequence.

12 . The method of claim 11 , wherein the guide RNA comprises a nucleotide sequence, selected from the group consisting of:

(SEQ ID NO: 415; sgRNA_368)

5′-UCCUAUAAGGUGUGAAAGUCUG-3′,

(SEQ ID NO: 416; sgRNA_369)

5′-UGAGCCCAUGCAGCUCUCCAGA-3′,

(SEQ ID NO: 417; sgRNA_370)

5′-CUCCUCAGUUGUGAGCCCAUGC-3′,

(SEQ ID NO: 418; sgRNA_371)

5′-GUAGAAGGGAUAUACAAAGUGG-3′,

(SEQ ID NO: 419; sgRNA_372)

5′-CCACUUUGUAUAUCCCUUCUAC-3′,

(SEQ ID NO: 420; sgRNA_373)

5′-GGUGUCUAUUUCCACUUUGUAU-3′,

and

(SEQ ID NO: 421; sgRNA_374)

5′-CAUGAGCAUGCAGAGGUGAGUA-3′;

or any of the aforementioned sequences wherein 1, 2, 3, 4, or 5 nucleotides is deleted from the 5′ and/or 3′ terminus of the nucleotide sequence.

13 . A method for treating amyloidosis in a subject, the method comprising administering to the subject a guide RNA and a polynucleotide encoding a base editor comprising a polynucleotide programmable DNA binding polypeptide and an adenosine or cytidine deaminase, wherein said guide RNA targets said base editor to effect an alteration of a nucleobase of the TTR polynucleotide sequence, wherein the adenosine deaminase domain comprises an arginine (R) or a threonine (T) at amino acid position 147 of the following amino acid sequence, and the adenosine deaminase domain has at least about 85% sequence identity to the following amino acid sequence

(SEQ ID NO: 4; TadA*7.10)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNR

AIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAM

IHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC

AALLCYFFRMPRQVFNAQKKAQSSTD,

wherein said guide RNA targets said fusion protein to effect an alteration of a nucleobase of the TTR polynucleotide sequence or

wherein the cytidine deaminase domain comprises an amino acid sequence with at least about 85% sequence identity to the amino acid sequence: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEV NFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNR QGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPC LNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 15), wherein said guide RNA targets said fusion protein to effect an alteration of a nucleobase of the TTR polynucleotide sequence.

14 . The method of claim 13 , wherein the altered nucleobase is