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 creating permanent changes to the genome that can result in at least one deletion, insertion, modulation, or inactivation of a transcriptional control sequence of a BCL11A gene in a cell by genome editing.
1. 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 deoxyribonucleic acid (DNA) endonucleases, a first single-molecule guide RNA (sgRNA) and a second sgRNA to effect two or more single-strand breaks (SSBs) or double-strand breaks (DSBs), within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene, that results in at least one of a permanent insertion, deletion, modulation, and an inactivation of a transcriptional control sequence of the BCL11A gene, wherein the one or more DNA endonucleases are Cas9 endonucleases, and wherein:
(i) the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 11 and the second sgRNA comprises the nucleic acid sequence of SEQ ID NO: 12, or
(ii) the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 11 and the second sgRNA comprises the nucleic acid sequence of SEQ ID NO: 13, or
(iii) the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 12 and the second sgRNA comprises the nucleic acid sequence of SEQ ID NO: 13.
2. The method of claim 1 , wherein the method comprises introducing into the human cell one or more polynucleotides encoding the one or more DNA endonucleases or wherein the method comprises introducing into the human cell one or more ribonucleic acids (RNAs) encoding the one or more DNA endonucleases, optionally wherein the one or more polynucleotides or the one or more RNAs is one or more modified polynucleotides or one or more modified RNAs.
3. The method of claim 1 , wherein the one or more DNA 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), optionally wherein the one or more DNA endonucleases each comprise two NLSs, one NLS located at the N-terminus and the second NLS located at the C-terminus and/or wherein the one or more NLSs is a SV40 NLS.
4. The method of claim 1 , wherein the one or more DNA endonucleases is pre-complexed with the first and/or second sgRNAs to form one or more ribonucleoproteins (RNPs), optionally wherein the weight ratio of sgRNAs to DNA endonuclease in the one or more RNPs is 1:1, further optionally wherein the DNA endonuclease is a S. pyogenes Cas9 comprising a N-terminus SV40 NLS and a C-terminus SV40 NLS, further optionally wherein the one or more RNPs is delivered to the cell by electroporation.
5. The method of claim 1 , wherein the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 11, the Cas9 endonuclease is a S. pyogenes Cas9 comprising a N-terminus SV40 NLS and a C-terminus SV40 NLS, wherein the weight ratio of the first and/or the second sgRNA to Cas9 endonuclease is 1:1 or wherein the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 12, the Cas9 endonuclease is a S. pyogenes Cas9 comprising a N-terminus SV40 NLS and a C-terminus SV40 NLS, wherein the weight ratio of sgRNA to Cas9 endonuclease is 1:1.
6. The method of claim 1 , wherein:
(a) an mRNA encoding the one or more Cas9 endonucleases and the first and/or second sgRNA are either each formulated into separate lipid nanoparticles or all co-formulated into a lipid nanoparticle, or
(b) an mRNA encoding the one or more Cas9 endonucleases is formulated into a lipid nanoparticle, and the first and/or second sgRNA are delivered to the cell by an adeno-associated virus (AAV) vector, or
(c) an mRNA encoding the one or more Cas9 endonucleases is formulated into a lipid nanoparticle, and the first and/or second sgRNA are delivered to the cell by electroporation.
7. An ex vivo method for treating a patient with a hemoglobinopathy, the method comprising:
(a) editing within or near a B-cell lymphoma 11A (BCL11A) gene or other DNA sequence that encodes a regulatory element of the BCL11A gene of an induced pluripotent stem cell (iPSC) or a mesenchymal stem cell, optionally wherein the mesenchymal stem cell is isolated from the patient's bone marrow or peripheral blood by aspiration of bone marrow and isolation of mesenchymal cells using density gradient centrifugation media;
(b) differentiating the genome-edited iPSC or mesenchymal stem cell into a hematopoietic progenitor cell, optionally wherein the differentiating step comprises one or more of the following to differentiate the genome-edited iPSC or mesenchymal stem cell into a hematopoietic progenitor cell: treatment with a combination of small molecules, delivery of master transcription factors, delivery of mRNA encoding master transcription factors, or delivery of mRNA encoding transcription factors; and
(c) implanting the hematopoietic progenitor cell into the patient, optionally wherein the implanting step comprises implanting the hematopoietic progenitor cell into the patient by transplantation, local injection, systemic infusion, or combinations thereof; and
wherein step (a) is performed by the method of claim 1 .
8. An ex vivo method for treating a patient with a hemoglobinopathy, the method comprising:
(a) editing within or near a B-cell lymphoma 11A (BCL11A) gene or other DNA sequence A that encodes a regulatory element of the BCL11A gene of a cell, optionally wherein the cell is a bone marrow cell, a hematopoietic progenitor cell, or a CD34+ cell; and
(b) implanting the genome-edited hematopoietic progenitor cell into the patient, optionally wherein the implanting step comprises implanting the genome-edited hematopoietic progenitor cell into the patient by transplantation, local injection, systemic infusion, or combinations thereof,
optionally further comprising: isolating the hematopoietic progenitor cell from the patient, optionally by treating the patient with granulocyte colony stimulating factor (GCSF) and/or 1,1′-(1,4-phenylenebismethylene)bis(1,4,8,11-tetraazacyclotetradecane) prior to the isolating step
further optionally wherein the hemoglobinopathy is selected from a group consisting of sickle cell anemia and thalassemia (α, β, δ, γ, and combinations thereof), and
wherein step (a) is performed by the method of claim 1 .
9. An in vivo method for treating a patient with a hemoglobinopathy, the method comprising: (a) editing a B-cell lymphoma 11A (BCL11A) gene in a cell of the patient, wherein step (a) is performed by the method of claim 1 , optionally wherein the hemoglobinopathy is selected from a group consisting of sickle cell anemia and thalassemia (α, β, δ, γ, and combinations thereof).
10. A kit comprising:
(i) a first gRNA comprising the nucleic acid sequence of SEQ ID NO: 11;
(ii) a second gRNA comprising the nucleic acid sequence of SEQ ID NO: 12; and
(iii) a third gRNA comprising the nucleic acid sequence of SEQ ID NO: 13,
optionally wherein (i), (ii), and/or (iii) is a modified gRNA and/or a sgRNA,
further optionally wherein the gRNAs in the kit are formulated in one composition.
11. The method of claim 1 , wherein the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 11 and the second sgRNA comprises the nucleic acid sequence of SEQ ID NO: 12.
12. The method of claim 1 , wherein the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 11 and the second sgRNA comprises the nucleic acid sequence of SEQ ID NO: 13.
13. The method of claim 1 , wherein the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 12 and the second sgRNA comprises the nucleic acid sequence of SEQ ID NO: 13.
14. A genetically engineered cell, which comprises genetic mutations, each of which is one of a permanent insertion, deletion, modulation, and an inactivation of a transcriptional control sequence of a BCL11A gene, wherein the genetic mutations occur at one or more sites targeted by a first sgRNA and a second sgRNA, wherein:
(i) the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 11 and the second sgRNA comprises the nucleic acid sequence of SEQ ID NO: 12, or
(ii) the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 11 and the second sgRNA comprises the nucleic acid sequence of SEQ ID NO: 13, or
(iii) the first sgRNA comprises the nucleic acid sequence of SEQ ID NO: 12 and the second sgRNA comprises the nucleic acid sequence of SEQ ID NO: 13,
optionally wherein the cell is a CD34+ human cell, further optionally a CD34+ human hematopoietic stem and progenitor cell.
15. The genetically engineered cell of claim 14 , wherein the cell exhibits a HbF mean percentage of HbF/(HbF+HbA) protein levels of at least 10%, optionally at least 15%, further optionally at least 20%, further optionally at least 25%, further optionally at least 30%, further optionally at least 40%, further optionally at least 50%.
16. A cell population comprising the genetically engineered cell of claim 14 , optionally wherein the population comprises cells having at least two different genetic mutations, further optionally wherein at least 70% of the population maintain multi-lineage potential for at least sixteen weeks after administration of the population to a subject; and/or wherein the population exhibits a mean allele editing frequency of 70% to 90%; and/or wherein the population exhibits a HbF mean percentage of HbF/(HbF+HbA) protein levels of at least 10%; and/or wherein the population exhibits an off-target indel rate of less than 1%.
17. A method for treating a patient with a hemoglobinopathy, comprising administering to a subject in need thereof an effective amount of the cell population of claim 16 , optionally wherein the hemoglobinopathy is selected from a group consisting of sickle cell anemia and thalassemia (α, β, δ, γ, and combinations thereof).