Lymphohematopoietic engineering using CAS9 base editors
Inventors
Moriarity, Branden • Webber, Beau • Lonetree, Cara-Lin • Diers, Miechaleen • Kluesner, Mitchell • Lahr, Walker • Pomeroy, Emily Joy
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Assignees
University of Minnesota System
Member
University of MinnesotaThe University of Minnesota's Research and Innovation Office (RIO) serves as a central resource for conducting, managing, and sponsoring research. It aims to facilitate collaborative research that addresses complex problems and promotes innovations that improve lives. RIO oversees research compliance and administration, fostering a culture of integrity and ethical practices in research. Additionally, RIO is committed to enhancing research capacity through various funding opportunities and strategic initiatives, including the management of specialized facilities for biocontainment research.
The University of Minnesota's Research and Innovation Office (RIO) serves as a central resource for conducting, managing, and sponsoring research. It aims to facilitate collaborative research that addresses complex problems and promotes innovations that improve lives. RIO oversees research compliance and administration, fostering a culture of integrity and ethical practices in research. Additionally, RIO is committed to enhancing research capacity through various funding opportunities and strategic initiatives, including the management of specialized facilities for biocontainment research.
Publication Number
US-12281327-B2
Publication Date
2025-04-22
Expiration Date
2039-03-13
Abstract
Provided herein are methods and systems for targeted gene disruption (knock-out, missense mutation) and targeted gene knock-in in mammalian cells using base editors and guide RNAs (gRNAs) designed to target splice acceptor-splice donor sites. Also provided herein are universally acceptable genetically engineered cells comprising targeted disruptions in immunotherapy-related genes and comprising a CAR/TCR for therapeutic applications.
Core Innovation
The invention provides methods and systems for targeted gene disruption, such as knock-out or missense mutation, and targeted gene knock-in in mammalian cells using base editors and guide RNAs (gRNAs) designed to target splice acceptor-splice donor sites. These methods enable controlled genome engineering by introducing a base editor fusion protein, which comprises a deaminase domain fused to a Cas9 nickase domain, and multiple splice acceptor-splice donor (SA-SD) gRNAs into lymphohematopoietic cells. The base editors employed include BE3, BE4, or adenine base editors (ABE).
The problem addressed by this invention is the inefficiency and safety concerns linked to conventional CRISPR-Cas9 approaches that induce double-stranded DNA breaks (DSBs) for multiplexed gene editing. Traditional methods, especially for precision alterations using homology-directed repair (HDR), are less efficient and can result in genotoxicity, including oncogenic chromosomal translocations. Therefore, more controlled and safer methods for multiplexed genetic engineering of human immune cells are needed, particularly methods that limit induction of toxic DSBs.
The core approach described in this invention enables efficient knock-out of genes by disrupting splice sites using SA-SD gRNAs, as well as introducing missense mutations or gene knock-ins by combining additional gRNAs and donor DNA templates. The methods apply to various lymphohematopoietic cells, including T cells, NK cells, B cells, and CD34+ HSPCs. Furthermore, the invention demonstrates that such methods permit efficient multiplex editing of clinically relevant genes, such as TRAC, B2M, and PDCD1, and simultaneous introduction of chimeric antigen receptors (CARs) or T cell receptors (TCRs) for the generation of universally acceptable genetically engineered cells for therapeutic applications.
Claims Coverage
The patent claims focus on a method for producing genetically engineered lymphohematopoietic cells using base editors and multiplexed splice acceptor-splice donor (SA-SD) gRNAs, with key inventive features centered on multiplex gene disruption and modification.
Multiplex gene disruption using splice acceptor-splice donor gRNAs and base editors
A method comprising introducing into a lymphohematopoietic cell: - A plasmid, mRNA, or protein encoding a base editor fusion protein with a deaminase domain fused to a Cas9 nickase domain that includes a base excision repair inhibitor domain. - Three or more splice acceptor-splice donor (SA-SD) gRNAs, wherein each targets a different nucleic acid sequence from SEQ ID NOs: 1-15, specifically including PDCD1, TRAC, and B2M genes. The method promotes disruption of splice sites targeted by these gRNAs, resulting in a genetically engineered cell with reduced expression of TRAC, B2M, and PDCD1 gene products compared to untransfected cells.
In summary, the independent claim broadly covers multiplexed, precise gene editing of lymphohematopoietic cells using base editors and specific SA-SD gRNAs targeting multiple immunologically relevant genes.
Stated Advantages
The methods allow controlled and safer multiplexed genetic engineering in human immune cells by limiting the induction of toxic double-stranded DNA breaks.
Splice acceptor-splice donor gRNA-mediated base editing achieves higher knock-out efficiency and lower rates of non-target editing and indel formation compared to conventional methods such as introduction of premature stop codons.
The techniques provide improved efficiency of gene knock-out, knock-in, or missense mutation in primary cells, such as T cells and CD34+ HSPCs, with reduced off-target effects.
The invention enables generation of universally acceptable, genetically engineered cells for therapeutic use without requiring cross-matching or immunosuppression.
Documented Applications
Production of genetically engineered T cells, NK cells, B cells, and CD34+ hematopoietic stem progenitor cells for studying cell biology and gene function.
Modeling diseases such as primary immunodeficiencies through targeted genome engineering in lymphohematopoietic cells.
Correction of disease-causing point mutations in patient immune cells.
Generation of novel cell products (e.g., T cells) equipped with chimeric antigen receptors (CAR) and/or T cell receptors (TCR) for therapeutic applications.
Engineering of universally acceptable, allogeneic immune cell therapeutics not limited by donor matching for immunotherapy.
Use in obtaining cleavage resistant Fc receptors in natural killer (NK) cells to enhance antibody-dependent cell-mediated cytotoxicity (ADCC).
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