Drug-resistant immune cells and methods of use thereof
Inventors
Osborn, Mark J. • Hippen, Keli • Blazer, Bruce R.
Assignees
University of Minnesota System
Publication Number
US-12343397-B2
Publication Date
2025-07-01
Expiration Date
2039-05-16
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Abstract
The present disclosure provides modified cells including pluripotent stem cells, hematopoietic precursor cells, and hematopoietic cells (e.g., modified Tregs) that are steroid and/or calcineurin inhibitor-resistant. The present disclosure provides methods for generating steroid and/or calcineurin inhibitor-resistant modified cells including pluripotent stem cells, hematopoietic precursor cells, and hematopoietic cells. Also provided herein are compositions and methods of treatment.
Core Innovation
The invention provides genetically modified regulatory T cells (Tregs) designed to resist immunosuppressive drugs such as steroids and calcineurin inhibitors. These modifications involve targeted gene editing that reduces or eliminates NR3C1 gene expression and the insertion of resistance genes against calcineurin inhibitors. The result is an enhanced Treg cell population with improved viability, persistence, and functionality in therapeutic settings. This innovation addresses the challenge of Treg susceptibility to standard immunosuppression, enabling more effective cell-based therapies for transplantation and autoimmune diseases.
The core innovation utilizes advanced gene editing techniques, such as CRISPR, to specifically disrupt NR3C1, which encodes the glucocorticoid receptor, thereby conferring steroid resistance. Concurrently, exogenous resistance genes for calcineurin inhibitors are inserted into the genome, allowing these cells to withstand immunosuppressant drugs. This dual modification enhances the therapeutic potential of Tregs, ensuring their survival and suppressive activity in immunosuppressed environments.
Claims Coverage
The claims describe methods for creating genetically modified Treg cells resistant to steroids and calcineurin inhibitors by disrupting NR3C1 and inserting resistance genes, using gene editing tools like CRISPR.
Genetic modification to downregulate NR3C1 expression
Introducing a gene editing system to target the NR3C1 gene locus, producing modifications such as indels or stop codons that reduce or eliminate gene expression, resulting in steroid resistance.
Insertion of exogenous calcineurin inhibitor resistance gene
Integrating a resistance gene into the Treg genome at the site of NR3C1 editing or elsewhere, conferring resistance to calcineurin inhibitors, with possible variants including genes like PPP3Ca, PPP3Cb, or encoding proteins such as CNa12 or CNb30.
Use of CRISPR systems for gene editing
Employing guide RNAs, such as SEQ ID NOs: 7-10, or base editors, to induce precise genetic modifications including indels or stop codons in NR3C1 for disruption.
Calcineurin inhibitor resistance gene variants
The resistance gene can be a mutant form of calcineurin encoding variants, including calcineurin A or B gene variants, or engineered proteins like CNa12, CNa22, or CNb30.
Generating modified cells for transplantation
Creating allogeneic Treg cells with these modifications for use in transplantation, autoimmune treatment, or disease management where immunosuppressive resistance is desired.
The claims cover methods for engineering Treg cells resistant to steroids and calcineurin inhibitors via gene editing—disrupting NR3C1 and inserting resistance genes—employing specific gRNAs and variants for therapeutic transplantation and autoimmune treatments.
Stated Advantages
Enhanced persistence and survival of modified immune cells in vivo even in the presence of immunosuppressive drugs such as steroids and calcineurin inhibitors.
Targeted gene disruption of NR3C1 provides specific resistance to glucocorticoids, preventing drug-induced impairment of Treg viability.
Insertion of calcineurin inhibitor resistance genes allows the modified cells to maintain activation and function despite immunosuppressive therapy, improving treatment efficacy.
Combining these modifications significantly enhances the therapeutic potential of Treg adoptive cell therapies in clinical settings like GVHD, autoimmune diseases, and transplantation.
Documented Applications
Use of steroid and calcineurin inhibitor-resistant Tregs for preventing and treating graft-versus-host disease (GVHD).
Application of genetically modified immune cells in transplantation to improve engraftment, tolerance, and immune regulation.
Treatment of autoimmune diseases such as Type 1 Diabetes using resistant regulatory T cells.
Enhancement of adoptive cell transfer therapies by ensuring Treg persistence and activity despite ongoing immunosuppressive regimens.
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