Therapeutic genome editing in Wiskott-Aldrich syndrome and x-linked thrombocytopenia
Described herein are systems and methods for treating, inhibiting, or ameliorating X-linked disorders including Wiskott-Aldrich Syndrome (WAS) and X-linked thrombocytopenia (XLT) in subjects that have been identified or selected as being ones that would benefit from a therapy to treat, inhibit, or ameliorate WAS or XLT. The systems include nuclease and vector donor constructs configured for co-delivery to modify endogenous WAS locus.
1 . A method of modifying a cell, comprising introducing into a cell:
(a) a Cas nuclease or a nucleic acid encoding the Cas nuclease, wherein the Cas nuclease is capable of cleaving a Wiskott-Aldrich Syndrome (WAS) locus in a cell genome;
(b) a guide RNA (gRNA), wherein the gRNA is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 31-34; and
(c) a vector comprising a WAS gene or portion thereof.
2 . The method of claim 1 , wherein the nuclease comprises a Cas9 nuclease.
3 . The method of claim 1 , wherein the vector is an adeno-associated viral (AAV) vector.
4 . The method of claim 1 , wherein the vector comprises a WAS cDNA.
5 . The method of claim 4 , wherein the vector lacks a promoter operably linked to the WAS cDNA.
6 . The method of claim 4 , wherein the WAS cDNA is inserted into a first exon of a WAS gene.
7 . The method of claim 1 , wherein the vector comprises an enhancer.
8 . The method of claim 1 , wherein the vector comprises a nucleic acid homologous to the WAS locus.
9 . The method of claim 1 , wherein the cell is selected from a T cell or a hematopoietic stem cell (HSC).
10 . The method of claim 9 , wherein the cell is a CD34+ HSC.
11 . The method of claim 1 , wherein the cell is obtained from a human male subject.
12 . A cell prepared by the method of claim 1 .
13 . A method of treating, inhibiting or ameliorating Wiskott-Aldrich syndrome (WAS) or X-linked thrombocytopenia (XLT) in a subject, comprising: administering the cell of claim 12 to the subject.
14 . The method of claim 13 , wherein the cell is autologous to the subject.
15 . The method of claim 1 , wherein the gRNA is encoded by the nucleotide sequence of SEQ ID NO: 31.
16 . The method of claim 1 , wherein the gRNA is encoded by the nucleotide sequence of SEQ ID NO: 32.
17 . The method of claim 1 , wherein the gRNA is encoded by the nucleotide sequence of SEQ ID NO: 33.
18 . The method of claim 1 , wherein the gRNA is encoded by the nucleotide sequence of SEQ ID NO: 34.
19 . The method of claim 13 , wherein the subject is male.