Sequence specific reagents targeting CCR5 in primary hematopoietic cells
Inventors
CATHOMEN, Toni • CORNU, Tatjana • Duchateau, Philippe • MUSSOLINO, Claudio • ROMITO, Marianna • Gouble, Agnes
Assignees
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Publication Number
US-11767512-B2
Publication Date
2023-09-26
Expiration Date
Abstract
The invention pertains to the field of cell therapy and HIV treatments. It provides with highly specific reagents for reducing or inactivating expression of CCR5 in primate and human primary cells, especially under the form of TALE-nucleases. These reagents allow the production of safer primary hematopoietic cells made resistant to HIV, stem cells or differentiated cells, for their infusion into HIV patients.
Core Innovation
The invention relates to a gene therapy/cell therapy for HIV that introduces and expresses CCR5-targeting sequence-specific reagents in primary hematopoietic cells. The approach reduces functional CCR5 expression by using a sequence-specific reagent that targets a genomic sequence encoding the N-terminal hydrophilic external region of the CCR5 protein. In particular, TALE-nucleases are used to bind target sequences selected from SEQ ID NO:5 and SEQ ID NO:6.
A central aspect is improved cleavage efficiency and specificity relative to prior CCR5 nucleases, including minimizing off-target CCR2 editing. The disclosure emphasizes that the sequence-specific reagent is expressed such that functional CCR5 is reduced by more than 50% in the resulting population of primary cells.
The document further describes formats and delivery/vector possibilities for expressing the TALE-nuclease sequence-specific reagent, including mRNA (transient expression) and other vehicle formats. It also outlines potential downstream engineering possibilities in CCR5-defective cells, including CAR expression and additional gene edits for safety/immune function, with extensive experimental and preclinical/functional context referenced in examples and figures.
Claims Coverage
The document provides two independent claims covering (i) a method for producing HIV resistant primary hematopoietic cells using a TALE-nuclease targeting the CCR5 N-terminal hydrophilic external region, and (ii) a TALE-nuclease monomer configured to bind CCR5-encoding genomic target sequences selected from SEQ ID NO:5 or SEQ ID NO:6 and cleave the corresponding CCR5-encoding sequence. The claim set includes multiple dependent refinements that introduce quantitative thresholds, specificity constraints, and construct/delivery format limitations.
Producing HIV resistant primary hematopoietic cells with CCR5 N-terminal targeting TALE-nuclease
Introducing into primary hematopoietic cells a nucleic acid encoding a sequence specific reagent targeting at least one genomic sequence encoding the N-terminal hydrophilic external region of the CCR5 protein, wherein the specific reagent is a TALE-nuclease that binds a target sequence selected from SEQ ID NO:5 and SEQ ID NO:6; and expressing the sequence specific reagent so that the expression of functional CCR5 is reduced by more than 50% in the resulting population of primary cells.
TALE-nuclease monomer for CCR5 N-terminal hydrophilic external region cleavage
A TALE-nuclease monomer designed to cleave a genomic sequence encoding the N-terminal hydrophilic external region of the CCR5 protein, wherein the monomer binds a target sequence of SEQ ID NO:5 or SEQ ID NO:6.
Overall, the claim coverage centers on TALE-nucleases that specifically bind genomic targets corresponding to the CCR5 N-terminal hydrophilic external region (SEQ ID NO:5/SEQ ID NO:6) and cleave to reduce functional CCR5 expression in primary hematopoietic cells, with dependent claims further adding quantitative CCR5 reduction and specificity constraints, including avoiding CCR2 gene cleavage, and specifying monomer/construct identity and format features.
Stated Advantages
Improved cleavage efficiency and specificity versus prior CCR5 nucleases.
Minimizing off-target CCR2 editing.
Reduction of functional CCR5 expression by more than 50% in the resulting population of primary cells.
Enabling HIV-resistant primary T-cells/HSPC for infusion.
Documented Applications
Producing HIV resistant primary hematopoietic cells for use in HIV-related therapy, including generation of HIV-resistant primary T-cells/HSPC for infusion.
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