Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 3 de 3
Filter
Add more filters










Database
Language
Publication year range
1.
Cancer Immunol Res ; 12(4): 462-477, 2024 Apr 02.
Article in English | MEDLINE | ID: mdl-38345397

ABSTRACT

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.


Subject(s)
Hematopoietic Stem Cell Transplantation , Multiple Myeloma , Humans , Multiple Myeloma/genetics , Multiple Myeloma/therapy , B-Cell Maturation Antigen/metabolism , HLA-E Antigens , T-Lymphocytes , Receptors, Antigen, T-Cell , Immunotherapy, Adoptive , Histocompatibility Antigens Class I/metabolism , Allografts/pathology
2.
Mol Cell ; 81(17): 3637-3649.e5, 2021 09 02.
Article in English | MEDLINE | ID: mdl-34478654

ABSTRACT

The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.


Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems/genetics , T-Lymphocytes/metabolism , CRISPR-Associated Protein 9/physiology , CRISPR-Associated Proteins/metabolism , CRISPR-Associated Proteins/physiology , DNA/genetics , Endonucleases/genetics , Gene Editing/methods , Genetic Techniques , Genome/genetics , Genomics/methods , Humans , Leukocytes, Mononuclear/metabolism , Molecular Conformation , RNA, Guide, Kinetoplastida/genetics , Structure-Activity Relationship , T-Lymphocytes/physiology
3.
Nat Biotechnol ; 37(12): 1471-1477, 2019 12.
Article in English | MEDLINE | ID: mdl-31740839

ABSTRACT

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.


Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Escherichia coli , Genome/genetics , HEK293 Cells , Humans , Models, Genetic
SELECTION OF CITATIONS
SEARCH DETAIL