Intracellular genomic transplant and methods of therapy
Inventors
Moriarity, Branden • Webber, Beau • McIvor, R. Scott • Choudhry, Modassir • Rosenberg, Steven A. • Palmer, Douglas C. • Restifo, Nicholas P.
Assignees
Intima Bioscience Inc • University of Minnesota System • US Department of Health and Human Services
Publication Number
US-10166255-B2
Publication Date
2019-01-01
Expiration Date
2036-07-29
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Abstract
Genetically modified compositions, such as non-viral vectors and T cells, for treating cancer are disclosed. Also disclosed are the methods of making and using the genetically modified compositions in treating cancer.
Core Innovation
The invention discloses genetically modified compositions, including non-viral vectors and T cells, for treating cancer by incorporating cancer-specific T cell receptors (TCRs) recognizing unique immunogenic mutations. These engineered cells comprise at least one gene disruption and at least one non-virally integrated TCR sequence, where the gene can be disrupted by the TCR insertion, often targeting immune checkpoint genes such as PD-1, CTLA-4, CISH, and others. The methods deliver exogenous TCR sequences into cells via non-viral means like CRISPR, TALEN, transposon-based systems, providing site-specific integration and disruption of endogenous genes.
The problem addressed is the limited success of immunotherapies, particularly in solid tumors due to the lack of identifiable tumor-specific molecules for targeted lymphocyte recognition and destruction. Current genetic engineering of lymphocytes has been largely effective only in hematologic tumors but fails to broadly apply to solid tumors. The disclosed methods innovate by utilizing non-viral gene editing to identify and integrate cancer-specific TCRs targeting tumor-associated neo-antigens defined by whole-exome sequencing, thus broadening immunotherapy to various cancer types.
The core technology enables the identification of cancer-specific TCRs from patient samples, their non-viral insertion into T cells via precise genome editing at specific loci, and simultaneous disruption of checkpoint genes to enhance T cell efficacy and viability. The approach improves genetic modification efficiency by homology-directed repair with the assistance of recombination arms and homologous recombination enhancers, while reducing cellular toxicity induced by exogenous nucleic acids through co-delivery of modifying compounds that inhibit cytosolic DNA sensing pathways.
Claims Coverage
The patent claims focus on methods and compositions for engineering primary cells, particularly T cells, by non-viral delivery of exogenous functional TCR or CAR sequences into endogenous genomic sites, especially the cytokine inducible SH2-containing protein (CISH) gene, using CRISPR or related endonucleases, combined with in situ reverse transcription of RNA templates.
Integration of exogenous functional TCR or CAR sequences into precise genomic loci
Introducing ribonucleic acid encoding exogenous functional T cell receptor (TCR) or chimeric antigen receptor (CAR) sequences into primary cells, followed by reverse transcription into DNA, and site-specific integration into genomic breaks within immune regulatory genes such as CISH mediated by endonucleases.
Use of non-viral genome editing tools
Employing endonucleases from systems including CRISPR, TALEN, Zinc Finger, transposon-based, ZEN, meganuclease, or Mega-TAL to introduce targeted double strand genomic breaks facilitating precise insertion of TCR or CAR sequences.
In situ reverse transcription of delivered RNA templates
Delivering RNA templates encoding exogenous antigen receptor sequences and reverse transcriptase to generate double stranded DNA in the nucleus for improved homologous recombination and integration efficiency.
Disruption of endogenous immune checkpoint and regulatory genes by targeted integration
Targeted introduction of exogenous receptor sequences into genes like CISH, PD-1, T cell receptor alpha and beta loci, resulting in simultaneous knockout or suppression of these genes to enhance anti-tumor T cell function.
Incorporation of nuclear localization and structural features in RNA templates
Engineering delivered RNA sequences to contain nuclear localization sequences and secondary structures such as hairpins to optimize nuclear import and reverse transcription efficiency.
The claims comprehensively cover compositions and methods for efficient, precise, non-viral genomic integration of exogenous TCR or CAR sequences into primary cells, particularly human T cells, with concurrent disruption of immune checkpoint genes like CISH to improve cell viability and therapeutic efficacy, employing reverse transcribed RNA templates and genome editing nucleases such as CRISPR.
Stated Advantages
Provides high efficiency gene transfer and expression of engineered T cell receptors into primary cells.
Increases cell survival rates by reducing cytotoxicity associated with exogenous nucleic acid delivery through modifying compounds.
Offers a process favoring homologous recombination over non-homologous end joining for precise genomic integration.
Enables expansion and recovery of modified immune cells with maintained immunologic and anti-tumor potency.
Documented Applications
Identification of cancer-specific TCRs recognizing unique immunogenic mutations for adoptive T cell transfer therapies targeting hematologic and solid tumors.
Treatment of cancer, including but not limited to bladder cancer, bone cancer, brain tumor, breast cancer, esophageal cancer, gastrointestinal cancer, leukemia, lymphoma, melanoma, myeloma, ovarian cancer, prostate cancer, sarcoma, stomach cancer, and thyroid cancer.
Engineering T cells with simultaneous checkpoint gene disruptions (e.g., PD-1, CTLA-4, CISH) for enhanced immunotherapy efficacy.
Use in autologous and allogeneic transplantation for cancer and disorders such as infections, autoimmune disorders, or graft-versus-host disease (GVHD).
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