Materials and methods for treatment of hemoglobinopathies
AssigneeVertex Pharmaceuticals Incorporated
InventorMatthew Hebden Porteus
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Abstract
The present application provides materials and methods for treating hemoglobinopathies. More specifically, the application provides methods for producing progenitor cells that are genetically modified via genome editing to increase the production of fetal hemoglobin (HbF), as well as modified progenitor cells (including, for example, CD34+ human hematopoietic stem cells) producing increased levels of HbF, and methods of using such cells for treating hemoglobinopathies such as sickle cell anemia and β-thalassemia.
Claims55
A method of increasing the level of fetal hemoglobin (HbF) in a human cell by genome editing using DNA endonuclease to effect a pair of double-strand breaks (DSBs), the first at a 5′ DSB locus and the second at a 3′ DSB locus within the δβ-globin region of human chromosome 11, causing a deletion or inversion of the chromosomal DNA between the 5′ DSB locus and the 3′ DSB locus that results in increased expression of γ-globin, thereby increasing the level of HbF in the cell.
A method of any of the preceding claims, wherein the method comprises introducing into the cell one or more polynucleotides encoding DNA endonuclease.
A method of any of the preceding claims, wherein the method comprises introducing into the cell one or more RNAs encoding DNA endonuclease.
A method of any of the preceding claims, wherein the DNA endonuclease is a Cas9 endonuclease and the method comprises introducing into the cell one or more polynucleotides encoding Cas9 and two guide RNAs, the first guide RNA comprising a spacer sequence that is complementary to a segment of the 5′ DSB locus, and the second guide RNA comprising a spacer sequence that is complementary to a segment of the 3′ DSB locus.
A method of any of the preceding claims, wherein the human cell is an isolated progenitor cell.
A method of any of the preceding claims, wherein there is a deletion of the chromosomal DNA between the 5′ DSB locus and the 3′ DSB locus.
A method of any of the preceding claims, wherein the 5′ DSB locus is proximal to the 5′ boundary of an HPFH deletion.
A method of any of the preceding claims, wherein the 5′ DSB locus is proximal to the 5′ boundary of an HPFH deletion selected from the group consisting of the HPFH-4 deletion, the HPFH-5 deletion, the HPFH-Kenya deletion, the HPFH-Black deletion, the long Corfu deletion, and the short Corfu deletion.
A method of any of the preceding claims, wherein the 3′ DSB locus is proximal to the 3′ boundary of an HPFH deletion.
A method of any of the preceding claims, wherein the 3′ DSB locus is proximal to the 3′ boundary of an HPFH deletion selected from the group consisting of the HPFH-4 deletion, the HPFH-5 deletion, the HPFH-Kenya deletion, the HPFH-Black deletion, the long Corfu deletion, and the short Corfu deletion.
A method of any of the preceding claims, wherein the 5′ DSB locus is proximal to the 5′ boundary and the 3′ locus is proximal to the 3′ boundary of an HPFH deletion.
A method of any of the preceding claims, wherein the 5′ DSB locus is proximal to the 5′ boundary and the 3′ locus is proximal to the 3′ boundary of an HPFH deletion selected from the group consisting of the HPFH-4 deletion, the HPFH-5 deletion, the HPFH-Kenya deletion, the HPFH-Black deletion, the long Corfu deletion, and the short Corfu deletion.
A method of any of the preceding claims, wherein the 3′ boundary of the deletion is proximal to Chr11:5224779 and the 5′ boundary of the deletion is proximal to Chr11:5237723.
A method of any of the preceding claims, wherein the 3′ boundary of the deletion is proximal to Chr11:5234665 and the 5′ boundary of the deletion is proximal to Chr11:5238138.
A method of any of the preceding claims, wherein the 3′ boundary of the deletion is proximal to Chr11:5233055 and the 5′ boundary of the deletion is proximal to Chr11:5240389.
A method of any of the preceding claims, wherein the 3′ boundary of the deletion is proximal to Chr11:5226631 and the 5′ boundary of the deletion is proximal to Chr11:5249422.
A method of any of the preceding claims, wherein the 3′ boundary of the deletion is proximal to Chr11:5196709 and the 5′ boundary of the deletion is proximal to Chr11:5239223.
A method of any of the preceding claims, wherein the 3′ boundary of the deletion is proximal to Chr11:5225700 and the 5′ boundary of the deletion is proximal to Chr11:5236750.
A method of any of the preceding claims, wherein there is a deletion of the chromosomal DNA between the 5′ DSB locus and the 3′ DSB locus.
A method of any of the preceding claims, wherein all or a portion of the δ-upstream region is deleted.
A method of any of the preceding claims, wherein the human cell is from a patient with a β-hemoglobinopathy which is a sickle cell disease or a β-thalassemia.
A method of any of the preceding claims, wherein all or a portion of the β-globin gene is deleted.
A method of increasing the level of fetal hemoglobin (HbF) in a human cell by genome editing using DNA endonuclease to effect a double-strand break (DSB) at one or more loci within the β-globin region of human chromosome 11, causing deletions or insertions of chromosomal DNA at the one or more loci that results in increased expression of γ-globin, thereby increasing the level of HbF in the cell.
A human cell produced by a method of any of the preceding claims.
Dependencies are inferred from the claim text.