CRISPR-CAS9 modified CD34+ human hematopoietic stem and progenitor cells and uses thereof
Provided herein, in some embodiments, are methods and compositions for treatment of subjects with β-thalassemia and subjects with severe sickle cell disease using autologous CRISPR-Cas9 modified CD34+ human hematopoietic stem and progenitor cells.
1. A composition comprising a single dose of CD34+ human hematopoietic stem and progenitor cells (hHSPCs) that comprise a genetic modification within a +58 DNase I hypersensitive site (DHS) within the erythroid lineage-specific enhancer of a human B-cell lymphoma 11A (BCL11A) gene (modified CD34+ hHSPCs), wherein the single dose comprises an effective amount to result in an absence of vaso-occlusive crises (VOCs) for at least 12 months after administration of the hHSPCs, wherein the genetic modification is produced by delivering to the CD34+ hHSPCs a Cas9 endonuclease and a guide RNA (gRNA) comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the single dose comprises a minimum dose of 3×10 6 CD34+ hHSPCs per kg.
2. The composition of claim 1 , further comprising a cryopreservation medium substantially free of serum, 5% dimethylsulfoxide (DMSO), and dextran-40.
3. The composition of claim 1 , wherein (i) the modified CD34+ hHSPCs exhibit an increase in γ/(γ+α)-globin mRNA ratios of 0.1 to 0.5 and/or wherein the modified CD34+ hHSPCs exhibit an increase in γ/(γ+β)-globin mRNA ratios of 0.2 to 0.6; (ii) the modified CD34+ hHSPCs comprise fetal hemoglobin (HbF) and adult hemoglobin (HbA) and exhibit a HbF mean percentage of HbF/(HbF+HbA) protein level of 15% to 50%; (iii) the modified CD34+ hHSPCs exhibit a mean allele editing frequency of 70% to 90%; (iv) at least 75% of the modified CD34+ hHSPCs maintain multi-lineage potential for at least sixteen weeks after administration of the modified CD34+ hHSPCs to a subject; (v) the modified CD34+ hHSPCs exhibit an on-target indel rate of at least 40%; and/or (vi) the modified CD34+ hHSPCs exhibit an off-target indel rate of less than 5%.
4. The composition of claim 1 , wherein the genetic modification is produced by delivering to the CD34+ hHSPCs a S. pyogenes Cas9 endonuclease or a variant thereof comprising a N-terminal SV40 nuclear localization signal (NLS).
5. The composition of claim 1 , wherein the genetic modification is produced by delivering to the CD34+ hHSPCs a Cas9 endonuclease and a guide RNA (gRNA) comprising the nucleotide sequence of SEQ ID NO: 1.
6. The composition of claim 1 , wherein the genetic modification is produced by delivering to the CD34+ hHSPCs a Cas9 endonuclease and a guide RNA (gRNA) comprising the nucleotide sequence of SEQ ID NO: 2.
7. A method comprising administering to a subject having a hemoglobinopathy a single dose of CD34+ human hematopoietic stem and progenitor cells (hHSPCs) that comprise a genetic modification within a +58 DNase I hypersensitive site (DHS) within the erythroid lineage-specific enhancer of a human B-cell lymphoma 11A (BCL11A) gene (modified CD34+ hHSPCs), wherein the single dose comprises an effective amount to result in an absence of vaso-occlusive crises (VOCs) for at least 12 months after administration of the hHSPCs, wherein the genetic modification is produced by delivering to the CD34+ hHSPCs a Cas9 endonuclease and a guide RNA (gRNA) comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the single dose comprises a minimum dose of 3×10 6 CD34+ hHSPCs per kg.
8. The method of claim 7 , further comprising administering red blood cells to the subject.
9. The method of claim 7 , further comprising administering busulfan to the subject.
10. The method of claim 9 , wherein the busulfan is administered prior to administration of the modified CD34+ hHSPCs.
11. The method of claim 10 , wherein the dose is adjusted based on pharmacokinetic level to achieve an area under the curve (AUC) of 4500 to 5500 μM/min.
12. The method of claim 7 , wherein
(i) the subject requires fewer blood transfusions within a two-year period of the time of administration of the modified CD34+ hHSPCs, or within a two-year period after the time of administration of the modified CD34+ hHSPCs, relative to a two-year period before the time of administration of the modified CD34+ hHSPCs;
(ii) the subject achieves transfusion reduction or transfusion independence for at least three months following administration of the modified CD34+ hHSPCs starting three months after administration of the modified CD34+ hHSPCs;
(iii) the subject achieves transfusion reduction or transfusion independence for at least six months following administration of the modified CD34+ hHSPCs starting three months after administration of the modified CD34+ hHSPCs;
(iv) the subject achieves transfusion reduction or transfusion independence for at least twelve months following administration of the modified CD34+ hHSPCs starting three months after administration of the modified CD34+ hHSPCs;
(v) the subject is no longer in need of iron chelation therapy within a 2 to 5 year period of the time of administration of the modified CD34+ hHSPCs, or within a 2 to 5 year period after the time of administration of the modified CD34+ hHSPCs, relative to a 2 to 5 year period before the time of administration of the modified CD34+ hHSPCs; and/or
(vi) the HbF level in the subject is at least 20% for at least three months starting at any time at or after the time of administration of the modified CD34+ hHSPCs.
13. The method of claim 7 , wherein (i) the modified CD34+ hHSPCs exhibit an increase in γ/(γ+α)-globin mRNA ratios of 0.1 to 0.5 and/or wherein the modified CD34+ hHSPCs exhibit an increase in γ/(γ+β)-globin mRNA ratios of 0.2 to 0.6; (ii) the modified CD34+ hHSPCs exhibit a HbF mean percentage of HbF/(HbF+HbA) protein levels of 15% to 50%; (iii) the modified CD34+ hHSPCs exhibit a ratio of (γ+β)/α-globin mRNA that is at or above 0.4; (iv) the modified CD34+ hHSPCs exhibit a mean allele editing frequency of 70% to 90%; (v) at least 50% of the modified CD34+ hHSPCs maintain multi-lineage potential for at least sixteen weeks after administration of the modified CD34+ hHSPCs; and/or (vi) the modified CD34+ hHSPCs exhibit an on-target indel rate of at least 40%.
14. The method of claim 7 , wherein the dose is 3×10 6 CD34+ hHSPCs per kg.
15. The method of claim 7 , wherein the genetic modification is produced by delivering to the CD34+ hHSPCs a Cas9 endonuclease and a guide RNA (gRNA) comprising the nucleotide sequence of SEQ ID NO: 1.
16. The method of claim 7 , wherein the genetic modification is produced by delivering to the CD34+ hHSPCs a Cas9 endonuclease and a guide RNA (gRNA) comprising the nucleotide sequence of SEQ ID NO: 2.