US2021024606A1PendingUtilityA1
Nuclease-mediated regulation of gene expression
Est. expiryMay 12, 2035(~8.8 yrs left)· nominal 20-yr term from priority
Inventors:Andreas Reik
A61P 7/00C12N 2510/00C12N 2501/20C12N 9/22C12N 5/0647A61P 43/00A61K 38/00C07K 14/705C07K 14/46C12N 9/224
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Claims
Abstract
The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic cell.
Claims
exact text as granted — not AI-modifiedWhat is claimed:
1 . A pharmaceutical composition comprising genetically modified cells, the genetically modified cells comprising cells comprising at least one zinc finger nuclease, the zinc finger nuclease comprising a zinc finger protein comprising 4, 5 or 6 fingers designated F1 to F4, F1 to F5 or F1 to F6, each finger comprising a recognition helix region that recognizes a target subsite wherein the protein is selected from the group consisting of
(i) a protein comprising the recognition helix regions as follows:
F1:
(SEQ ID NO: 7)
STGNLTN;
F2:
(SEQ ID NO: 5)
TSGSLTR;
F3:
(SEQ ID NO: 2)
DQSNLRA;
and
F4:
(SEQ ID NO: 6)
AQCCLFH;
(ii) a protein comprising the recognition helix regions as follows:
F1:
(SEQ ID NO: 2)
DQSNLRA;
F2:
(SEQ ID NO: 3)
RPYTLRL;
F3:
(SEQ ID NO: 8)
SRGALKT;
F4:
(SEQ ID NO: 5)
TSGSLTR;
F5:
(SEQ ID NO: 2)
DQSNLRA;
and
F6:
(SEQ ID NO: 6)
AQCCLFH;
(iii) a protein comprising the recognition helix regions as follows:
F1:
(SEQ ID NO: 2)
DQSNLRA;
F2:
(SEQ ID NO: 9)
RNFSLTM;
F3:
(SEQ ID NO: 10)
SNGNLRN
or
(SEQ ID NO: 11)
SSYNLAN;
F4:
(SEQ ID NO: 5)
TSGSLTR;
F5:
(SEQ ID NO: 2)
DQSNLRA;
and
F6:
(SEQ ID NO: 6)
AQCCLFH;
(iv) a protein comprising the recognition helix regions as follows:
F1:
(SEQ ID NO: 13)
RSDHLTQ;
F2:
(SEQ ID NO: 14)
QSGHLAR;
F3:
(SEQ ID NO: 15)
QKGTLGE;
F4:
(SEQ ID NO: 16)
RHRDLSR;
and
F5:
(SEQ ID NO: 17)
RRDNLHS;
(v) a protein comprising the recognition helix regions as follows:
F1:
(SEQ ID NO: 19)
RNDHRTT;
F2:
(SEQ ID NO: 20)
QKAHLIR;
F3:
(SEQ ID NO: 15)
QKGTLGE;
F4:
(SEQ ID NO: 25)
LKRTLKR;
and
F5:
(SEQ ID NO: 17)
RRDNLHS;
(vi) a protein comprising the recognition helix regions as follows:
F1:
(SEQ ID NO: 13)
RSDHLTQ;
F2:
(SEQ ID NO: 22)
QRAHLTR;
F3:
(SEQ ID NO: 15)
QKGTLGE
or
(SEQ ID NO: 24)
QSGTRNH;
F4:
(SEQ ID NO: 23)
HRNTLVR;
and
F5:
(SEQ ID NO: 17)
RRDNLHS;
(vii) a protein comprising the recognition helix regions as follows:
F1:
(SEQ ID NO: 13)
RSDHLTQ;
F2:
(SEQ ID NO: 20)
QKAHLIR;
F3:
(SEQ ID NO: 15)
QKGTLGE
or
(SEQ ID NO: 24)
QSGTRNH;
F4:
(SEQ ID NO: 21)
RGRDLSR;
and
F5:
(SEQ ID NO: 17)
RRDNLHS;
(viii) a protein comprising the recognition helix regions as follows:
F1:
(SEQ ID NO: 13)
RSDHLTQ;
F2:
(SEQ ID NO: 14)
QSGHLAR;
F3:
(SEQ ID NO: 24)
QSGTRNH;
F4:
(SEQ ID NO: 16)
QSSDLSR;
and
F5:
(SEQ ID NO: 17)
RRDNLHS.
2 . The pharmaceutical composition of claim 1 , wherein the cells comprise one or more polynucleotides encoding the zinc finger nuclease.
3 . The pharmaceutical composition of claim 1 , wherein the genetically modified cells are hematopoietic stem cells or erythroid precursor cells.
4 . The pharmaceutical composition of claim 3 , wherein the cells are human cells.
5 . The pharmaceutical composition of claim 1 , wherein the genetic modification is selected from the group consisting of insertions, deletions and combinations thereof.
6 . The pharmaceutical composition of claim 1 , wherein the genetic modification is within the +58 region of the BCL11A enhancer sequence.
7 . The pharmaceutical composition of claim 1 , further comprising genetically modified progeny cells produced from the genetically modified cells.
8 . The pharmaceutical composition of claim 7 , wherein the genetically modified progeny cells comprise red blood cell (RBC) precursor cells or RBCs.
9 . The pharmaceutical composition of claim 7 , wherein the genetically modified cells exhibit increased expression of gamma and/or beta globin as compared to cells without the genomic modification.
10 . The pharmaceutical composition of claim 1 , wherein the zinc finger nuclease comprises a pair of zinc finger nucleases.
11 . The pharmaceutical composition of claim 1 , wherein the pair of zinc finger nucleases comprise left and right zinc finger nucleases.
12 . The pharmaceutical composition of claim 11 , wherein the left zinc finger nuclease comprises the zinc finger protein of (i), (ii) or (iii) and the right zinc finger nuclease comprises the zinc finger protein of (iv), (v), (vi), (vii) or (viii).
13 . The pharmaceutical composition of claim 1 , wherein the genetic modification comprises an insertion.
14 . The pharmaceutical composition of claim 13 , wherein an exogenous sequence is inserted into the genome of the cells.
15 . The pharmaceutical composition of claim 1 , wherein the genetic modification comprises a deletion.
16 . A method of increasing globin production in a subject, the method comprising:
administering the pharmaceutical composition of claim 7 to the subject.
17 . The method of claim 16 , wherein the subject is a human.
18 . The method of claim 17 , wherein the pharmaceutical composition is administered to bone marrow of the subject and wherein the cell engrafts, differentiates and matures in the subject.
19 . The method of claim 16 , wherein the subject has a hemoglobinopathy.
20 . The method of claim 19 , wherein the hemoglobinopathy is a beta-thalassemia or sickle cell disease.
21 . A kit comprising the pharmaceutical composition of claim 7 .Join the waitlist — get patent alerts
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