Regulation of gene expression using engineered nucleases
Inventors
Miller, Jeffrey C. • Rebar, Edward J.
Assignees
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Publication Number
US-11845965-B2
Publication Date
2023-12-19
Expiration Date
Abstract
The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a hematopoietic cell.
Core Innovation
The invention relates to genome-engineering strategies to modulate BCL11A expression by generating a genomic modification in the BCL11A locus. The disclosed targeting focuses on the BCL11A enhancer, including the +58 region, in hematopoietic stem/progenitor cells, for increasing gamma (HbF) expression and modulating globin expression.
The disclosed work relates to a genetically modified cell with a genomic modification in the BCL11A locus and a zinc finger nuclease (ZFN) comprising left and right ZFNs. The left ZFN comprises the amino acid sequence of SEQ ID NO:29 and the right ZFN comprises the amino acid sequence of SEQ ID NO:31, directed to the BCL11A enhancer.
The disclosure provides experimental support for reduced off-target cleavage and functional globin modulation outcomes. It reports deep-sequencing-based analysis and off-target mapping, reduced off-target indels while retaining on-target activity, and in vitro erythroid differentiation showing maintained BCL11A enhancer modification and increased gamma globin mRNA and protein, with early fetal hemoglobin protein increase and reported fetal globin protein percentages.
Claims Coverage
The independently claimed subject matter is a genetically modified cell containing a genomic modification in the BCL11A locus together with a ZFN having left and right ZFNs defined by SEQ ID NO:29 and SEQ ID NO:31. The inventive features are further refined by additional ZFN-encoding polynucleotides, narrowing to CD34+ hematopoietic stem cells, and extending to a pharmaceutical composition and a method of increasing globin production in a subject, including beta-thalassemia or sickle cell disease.
Genetic modification in the BCL11A locus with defined ZFN amino acid sequences
A genetically modified cell comprising a genomic modification in the BCL11A locus and further comprising a zinc finger nuclease (ZFN) with a left ZFN having the amino acid sequence of SEQ ID NO:29 and a right ZFN having the amino acid sequence of SEQ ID NO:31.
Additional polynucleotides encoding ZFN components
The genetically modified cell further comprising polynucleotides encoding additional zinc-finger nuclease parts, where a polynucleotide encodes the left zinc-finger nuclease of SEQ ID NO:28 and a polynucleotide encodes the right zinc-finger nuclease of SEQ ID NO:30.
CD34+ hematopoietic stem cell
The genetically modified cell is a CD34+ hematopoietic stem cell.
Pharmaceutical composition containing the genetically modified cells
A pharmaceutical composition comprising the genetically modified cells.
Method of increasing globin production
A method for increasing globin production in a subject by administering the pharmaceutical composition.
Hemoglobinopathy limited to beta-thalassemia or sickle cell disease
The subject's hemoglobinopathy is beta-thalassemia or sickle cell disease.
Overall claim coverage centers on genetically modified CD34+ hematopoietic stem cells with a BCL11A locus genomic modification mediated by a specific ZFN left/right amino acid pair, further defined by additional ZFN polynucleotides and extended to pharmaceutical compositions and administration-based methods for increasing globin production in subjects with beta-thalassemia or sickle cell disease.
Stated Advantages
Increased gamma (HbF) expression for modulating globin expression.
Reduced off-target cleavage, including reduced off-target indels while maintaining on-target activity.
Documented Applications
Deep-sequencing-based validation of ZFN activity and specificity in human CD34+ HSPCs, including off-target indel quantification and on-target indel activity at the BCL11A enhancer.
In vitro erythroid differentiation to assess BCL11A enhancer modification retention after freeze/thaw and differentiation, along with increased gamma globin mRNA and protein and early fetal hemoglobin protein increase.
Mouse NSG engraftment and downstream assessment of gene modification retention and fetal globin elevation in erythroid progeny, including human CD45 chimerism assessment.
Modulation of globin expression by increasing gamma (HbF) expression in hematopoietic stem/progenitor cells for hemoglobinopathy treatment, including beta-thalassemia and sickle cell disease.
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