MATERIALS AND METHODS FOR TREATMENT OF HEMOGLOBINOPATHIES
Materials and methods for treating a patient with a hemoglobinopathy, both ex vivo and in vivo, and materials and methods for deleting, modulating, or inactivating a transcriptional control sequence of a BCL11A gene in a cell by genome editing.
1 .- 93 . (canceled)
94 . A genetically engineered cell, which is produced by a method comprising:
introducing into a human cell one or more S. pyogenes Cas9 endonucleases and one or more guide RNAs (gRNAs) to effect one or more double-strand breaks (DSBs) within or near a B-cell lymphoma 11A (BCL11A) gene, that results in a permanent deletion or inactivation of the BCL11A gene,
wherein the one or more gRNAs comprise a single molecule gRNA (sgRNA) comprising the nucleic acid sequence of SEQ ID NO: 71,959.
95 . A genetically engineered cell, which comprises a genetic mutation, which is one of a permanent deletion or inactivation of a transcriptional control sequence of a B-cell lymphoma 11A (BCL11A) gene, wherein the genetic mutation occurs at the site targeted by a single molecule gRNA (sgRNA) comprising the nucleic acid sequence of SEQ ID NO: 71,959.
96 . The genetically engineered cell of claim 95 , wherein the cell is a CD34 + human cell.
97 . A population of genetically engineered cells, comprising the genetically engineered cell of claim 95 .
98 . A single molecule guide ribonucleic acid (sgRNA) comprising the nucleic acid sequence of SEQ ID NO: 71,959.
99 . A method for editing a B-cell lymphoma 11A (BCL11A) gene in a human cell by genome editing, the method comprising:
introducing into the human cell one or more Cas9 endonucleases and one or more gRNAs to effect one or more double-strand breaks (DSBs) within or near the BCL11A gene, that results in a permanent deletion or inactivation of the BCL11A gene,
wherein the one or more gRNAs comprise the sgRNA of claim 98 .
100 . The method of claim 99 , wherein the method comprises introducing into the human cell one or more polynucleotides encoding the one or more Cas9 endonucleases.
101 . The method of claim 100 , wherein the method comprises introducing into the human cell one or more ribonucleic acids (RNAs) encoding the one or more Cas9 endonucleases.
102 . The method of claim 99 , wherein the one or more Cas9 endonucleases each comprise, at the N-terminus, the C-terminus, or both the N-terminus and C-terminus, one or more nuclear localization signals (NLSs).
103 . The method of claim 99 , wherein the one or more Cas9 endonucleases each comprise two NLSs, one NLS located at the N-terminus and the second NLS located at the C-terminus.
104 . The method of claim 103 , wherein the one or more NLSs is a SV40 NLS.
105 . The method of claim 99 , wherein the one or more Cas9 endonucleases is pre-complexed with one or more gRNAs to form one or more ribonucleoproteins (RNPs).
106 . The method of claim 105 , wherein the weight ratio of gRNA to Cas9 endonuclease in the RNP is 1:1.
107 . The method of claim 105 , wherein the one or more RNPs is delivered to the human cell by electroporation.
108 . The method of claim 99 , wherein the one or more Cas9 endonucleases is a S. pyogenes Cas9 comprising a N-terminus SV40 NLS and a C-terminus SV40 NLS, and wherein the weight ratio of gRNA to Cas9 endonuclease is 1:1.
109 . An ex vivo method for treating a patient with a hemoglobinopathy, the method comprising:
(a) isolating a CD34 + hematopoietic stem or progenitor cell (HSPC) from the patient;
(b) editing within or near a B-cell lymphoma 11A (BCL11A) gene of the CD34 + HSPC; and
(c) implanting the genome-edited CD34 + HSPC into the patient, wherein step (b) is performed by the method of claim 99 .
110 . The method of claim 109 , wherein in step (b), the one or more Cas9 endonucleases is a S. pyogenes Cas9 comprising a N-terminus SV40 NLS and a C-terminus SV40 NLS, and wherein the weight ratio of gRNA to Cas9 endonuclease is 1:1.
111 . The method of claim 109 , wherein the method further comprises treating the patient with granulocyte colony stimulating factor (GCSF) prior to the isolating step.
112 . The method of claim 111 , wherein the treating step is performed in combination with Plerixaflor.
113 . The method of claim 109 , wherein the implanting step comprises implanting the genome-edited CD34 + HSPC into the patient by transplantation, local injection, systemic infusion, or combinations thereof.
114 . The method of claim 109 , wherein the hemoglobinopathy is selected from a group consisting of sickle cell disease and thalassemia.
115 . The method of claim 114 , wherein the hemoglobinopathy is a thalassemia and the thalassemia is selected from the group consisting of α, β, δ, γ, and combinations thereof.
116 . The method of claim 115 , wherein the thalassemia is β-thalassemia.