Method of inactivating a glucocorticoid receptor gene in an isolated cell
Inventors
Reik, Andreas • Jensen, Michael • Holmes, Michael C. • Gregory, Philip D. • Ando, Dale
Assignees
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Publication Number
US-11884930-B2
Publication Date
2024-01-30
Expiration Date
Abstract
Disclosed herein are methods and compositions for inactivation of the human glucocorticoid receptor (GR) gene by targeted cleavage of genomic DNA encoding the GR. Such methods and compositions are useful, for example, in therapeutic applications which require retention of immune function during glucocorticoid treatment.
Core Innovation
The patent describes a method of selecting isolated human cells by integrating a transgene into an endogenous glucocorticoid receptor (GR) gene using zinc finger nuclease (ZFN) activity. The approach introduces one or more polynucleotides encoding a first and second pair of ZFN and a transgene polynucleotide flanked by homology arms that target a human glucocorticoid receptor (GR) gene, such that the transgene is integrated into an endogenous GR gene. After introduction, the isolated human cells are contacted with a corticosteroid so that cells not comprising the integrated transgene are killed while cells with transgene integration are selected.
The ZFNs are engineered for targeted cleavage at the human GR gene locus, and the cleavage can produce NHEJ-mediated mutations at the targeted GR genomic location, including frameshifts and indels that result in GR loss of function. The patent links GR gene disruption to nonsense-mediated decay and persistent glucocorticoid resistance or GR-negative behavior in the edited cells. In addition, targeted donor-mediated integration of an exogenous sequence is described as enabling permanent GR disruption, including embodiments involving a zetakine transgene for selection.
The patent further describes specific engineered GR-targeted ZFN recognition sequences and obligate heterodimer cleavage half-domains to reduce aberrant cleavage. It includes exemplary zinc finger recognition-helices sets and engineered FokI cleavage half-domain pairing (E490K:I538K and Q486E:I499L) to support specificity. The documented rationale ties the GR-negative, glucocorticoid-resistant edited cells to immunotherapy under corticosteroid conditions, with embodiments using viral delivery vectors including replication-defective adenovirus, hybrid adenovirus, and non-integrating lentivirus.
Claims Coverage
The partial content provides one independent claim covering a selection method using GR-targeted ZFNs and corticosteroid kill/selection, including inventive specificity from defined zinc finger recognition-helices sets. Dependent claims refine recognition-helices specificity, narrow the corticosteroid to dexamethasone, and restrict nucleic-acid delivery to particular viral vector types and replication-defective delivery.
Corticosteroid selection of transgene-integrated GR cells
Introducing (i) one or more polynucleotides encoding a first and second pair of zinc finger nuclease (ZFN) and (ii) a polynucleotide comprising a transgene flanked by homology arms that target a human glucocorticoid receptor (GR) gene into isolated human cells such that the transgene is integrated into an endogenous GR gene, and contacting the isolated human cells with a corticosteroid to kill cells not comprising the integrated transgene and select cells comprising the transgene integrated into the endogenous GR gene.
Engineered GR-targeting ZFN recognition-helices sets for integration
The method wherein the first ZFN comprises a zinc finger DNA-binding domain comprising specified recognition helices in a specified order and the second ZFN comprises a zinc finger DNA-binding domain comprising specified recognition helices in a specified order, or alternative specified recognition-helices sets for the first and second ZFNs.
Across the provided claim set, the inventive core is GR-locus integration of a transgene using a pair of engineered ZFNs followed by corticosteroid kill/selection, with claim refinements based on defined ZFN zinc finger recognition-helices sets and narrowing to particular corticosteroid and delivery-vector embodiments.
Stated Advantages
Enables selection of isolated human cells with a transgene integrated into an endogenous glucocorticoid receptor (GR) gene by killing cells not comprising the integrated transgene upon corticosteroid contacting.
Allows maintenance of anti-tumor functions under corticosteroid treatment by generating glucocorticoid-resistant/GR-negative engineered T cells.
Documented Applications
Immunotherapy rationale using engineered T cells that remain anti-tumor under corticosteroid treatment by generating glucocorticoid-resistant/GR-negative engineered cells.
A selection use case for isolating human cells in which a transgene is integrated into an endogenous GR gene, using ZFN-mediated integration followed by corticosteroid kill/selection.
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