US2023067653A1PendingUtilityA1
Engineered target specific nucleases
Est. expiryFeb 8, 2038(~11.6 yrs left)· nominal 20-yr term from priority
C12N 2800/80C12N 2750/14143A61K 38/465C12N 15/907C12N 15/86C12N 9/16A61K 48/00C12N 9/644C12N 9/2402C12Y 302/01076C12Y 301/06013A61K 38/47C12N 2750/14343A61P 7/00A61K 38/4846C12N 7/00A61K 48/0066C12Y 304/21022C12N 2310/20C12N 9/22
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Claims
Abstract
Described herein are engineered nucleases comprising mutations in the cleavage domain (e.g., FokI or homologue thereof) and/or DNA binding domain (zinc finger protein, TALE, single guide RNA) such that on-target specificity is increased.
Claims
exact text as granted — not AI-modified1 - 35 . (canceled)
36 . An isolated polynucleotide encoding a Zinc Finger Nuclease (ZFN) and at least one of:
(a) a polynucleotide encoding an exogenous peptide; (b) a modified 3′ UTR; and/or (c) a 5′ UTR.
37 . The polynucleotide of claim 36 , wherein the polynucleotide comprises the polynucleotide encoding an exogenous peptide, the modified 3′ UTR, and the 5′ UTR.
38 . The polynucleotide of claim 36 , wherein the sequence encoding the exogenous peptide encodes a polyhistidine tag.
39 . The polynucleotide of claim 36 , wherein the polynucleotide encoding the exogenous peptide encodes a cationic peptide.
40 . The polynucleotide of claim 39 , wherein the cationic peptide comprises a Flag tag.
41 . The polynucleotide of claim 40 , wherein the Flag tag is a 3×Flag tag.
42 . The polynucleotide of claim 41 , wherein the 3×Flag tag comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 71.
43 . The polynucleotide of claim 36 , wherein the modified 3′ UTR comprises a WPRE.
44 . The polynucleotide of claim 43 , wherein the WPRE comprises one of: SEQ ID NO: 42, SEQ ID NO: 53, SEQ ID NO: 68, or SEQ ID NO: 69.
45 . The polynucleotide of claim 36 , wherein the 5′ UTR comprises a beta-globin 5′ UTR.
46 . The polynucleotide of claim 45 , wherein the beta-globin 5′ UTR is a Xenopus beta-globin 5′ UTR.
47 . The polynucleotide of claim 46 , wherein the Xenopus beta-globin 5′ UTR comprises SEQ ID NO: 1.
48 . The polynucleotide of claim 36 , wherein the polynucleotide encodes a ZFN comprising 6 zinc finger domains each comprising recognition helix regions F1 to F5 or F1 to F6 of the ZFNs arranged in the order of F1 to F5 or F1 to F6 as shown in a single row of the table below:
F1
F2
F3
F4
F5
F6
QSGNLSR
LKQNLCM
WADNLQN
TSGNLTR
RQSHLCL
N/A
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 14)
NO: 15)
NO: 16)
NO: 17)
NO: 18)
QSGNLAR
LKQNLCM
WQSNLQN
TSGNLTR
RRSHLTS
N/A
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 19)
NO: 15)
NO: 20)
NO: 17)
NO: 21)
QSGNLAR
LKQNLCM
LITTLRN
TSGNLTR
RQSHLCL
N/A
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 19)
NO: 15)
NO: 22)
NO: 17)
NO: 18)
QSGNLAR
LKQNLCM
WASNLQN
TSGNLTR
RQSHLCL
N/A
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 19)
NO: 15)
NO: 23)
NO: 17)
NO: 18)
TPQLLDR
LKHNLLT
DQSNLNA
RNFSLTM
LRHDLDR
HRSNLNK
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 24)
NO: 25)
NO: 26)
NO: 27)
NO: 28)
NO: 29)
QSSDLSR
LKHNLLT
DQSNLRA
RNFSLTM
LRHDLER
HRSNLNK
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 30)
NO: 25)
NO: 31)
NO: 27)
NO: 32)
NO: 29)
TPQLLDR
LKWNLRT
DQSNLNA
RNFSLTM
LRHDLDR
HRSNLNK
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 24)
NO: 33)
NO: 26)
NO: 27)
NO: 28)
NO: 29)
49 . The polynucleotide of claim 48 , wherein the polynucleotide encoding an exogenous peptide comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 71, the modified 3′ UTR comprises one of: SEQ ID NO: 42, SEQ ID NO: 53, SEQ ID NO: 68, or SEQ ID NO: 69, and the 5′ UTR comprises SEQ ID NO: 1.
50 . A method for improving efficiency of cleavage and/or activity of a Zinc Finger Nuclease (ZFN) comprising modifying a polynucleotide encoding the ZFN by incorporation of the polypeptide of claim 36 .
51 . The method of claim 50 , wherein each of polynucleotide comprises the polynucleotide of claim 37 .
52 . The method of claim 50 , wherein the activity of the ZFN encoded by a modified polynucleotide is at least about 2-fold more specific than a ZFN encoded by an unmodified polynucleotide.
53 . The method of claim 50 , wherein the activity of the ZFN encoded by a modified polynucleotide results in at least about 2-fold more integration of a transgene than a ZFN encoded by an unmodified polynucleotide.
54 . The method of claim 50 , wherein the efficiency of cleavage of the ZFN encoded by a modified polynucleotide is at least about 2-fold more efficient than a ZFN encoded by an unmodified polynucleotide.
55 . The method of claim 50 , wherein the expression level of the ZFN encoded by a modified polynucleotide is at least about 2-fold higher than the expression level of a ZFN encoded by an unmodified polynucleotide.Cited by (0)
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