Differential proliferation of human hematopoietic stem and progenitor cells using truncated erythropoietin receptors
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsMatthew H. Porteus, Michael Kyle Cromer, Joab Camarena
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Abstract
Edited cell chimerism is currently one of the greatest bottlenecks to clinical efficacy of gene therapies for the hemoglobinopathies. For example, it is difficult to go from low hematopoietic stem cell (HSC) edited cell chimerism in the bone marrow to high edited red blood cell (RBC) chimerism in the bloodstream. The present disclosure provides methods and compositions for genetically modifying hematopoietic stem and progenitor cells (HSPCs), in particular by creating HSPCs that express truncated forms of the EPO.receptor (tEPOR).
Claims88
A method of genetically modifying a hematopoietic stem and progenitor cell (HSPC), the method comprising:
- introducing into the HSPC an RNA-guided nuclease and a guide RNA that specifically targets a sequence within the cytoplasmic domain-encoding region of the erythropoietin receptor (EPOR) locus in the genome of the cell;
- wherein
- the RNA-guided nuclease cleaves the EPOR locus in the genome of the cell, resulting in the expression of a truncated erythropoietin receptor (tEPOR) in the cell and thereby generating a genetically modified HSPC;
- and wherein
- the expression of the tEPOR increases the sensitivity of the cell to erythropoietin (EPO) and/or increases the proliferation of the cell in the presence of EPO as compared to the sensitivity and/or proliferation of a non-genetically modified HSPC.
A method of genetically modifying a hematopoietic stem and progenitor cell (HSPC), the method comprising:
- introducing into the HSPC an RNA-guided nuclease, a donor template comprising a transgene encoding a truncated erythropoictin receptor (tEPOR), and a guide RNA that specifically targets a sequence within a safe harbor locus in the genome of the cell;
- wherein
- the donor template comprises a first homologous region comprising complementarity to the safe harbor locus upstream of the guide RNA target site, a second homologous region comprising complementarity to the safe harbor locus downstream of the guide RNA target site, wherein the first and second homology regions flank the tEPOR transgene on the template, wherein
- the RNA-guided nuclease cleaves the safe harbor locus in the genome of the cell and the transgene is integrated into the genome at the cleaved safe harbor locus, thereby generating a genetically modified HSPC;
- and wherein
- the integrated transgene results in expression of the tEPOR in the genetically modified HSPC.
A donor template comprising a homology region comprising SEQ ID NO:
- 1 or SEQ ID NO:
- 2 or a subsequence thereof, or a nucleotide sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:
- 1 or SEQ ID NO:
- 2 or a subsequence thereof.
A donor template comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:
- 3, 6, and 9 and subsequences thereof, or a nucleotide sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NOS:
- 3, 6, or 9, or a subsequence thereof.
A transgene comprising a nucleotide sequence encoding a tEPOR, wherein the nucleotide sequence comprises the sequence of SEQ ID NO:10, or a nucleotide sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:10.
A guide RNA comprising a target sequence comprising SEQ ID NO:
- 11 or SEQ ID NO:12, or a sequence comprising 1, 2, or 3 mismatches with SEQ ID NO:
- 11 or SEQ ID NO:12.
Dependencies are inferred from the claim text.