Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of ß2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness.

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

Human CD19-specific switchable CAR T-cells are efficacious as constitutively active CAR T-cells but cause less morbidity in a mouse model of human CD19+ malignancy.

Current Food and Drug Administration (FDA)-approved CD19-specific chimeric antigen receptor (CAR) T-cell therapies for B-cell malignancies are constitutively active and while efficacious, can cause morbidity and mortality. Their toxicities might be reduced if CAR T-cell activity was regulatable rather than constitutive. To test this, we compared the efficacies and morbidities of constitutively active (conventional) and regulatable (switchable) CAR (sCAR) T-cells specific for human CD19 (huCD19) in an immune-competent huCD19+ transgenic mouse model.Conventional CAR (CAR19) and sCAR T-cells were generated by retrovirally transducing C57BL/6 (B6) congenic T-cells with constructs encoding antibody-derived single chain Fv (sFv) fragments specific for huCD19 or a peptide neoepitope (PNE), respectively. Transduced T-cells were adoptively transferred into huCD19 transgenic hemizygous (huCD19Tg/0 ) B6 mice; healthy B-cells in these mice expressed huCD19Tg Prior to transfer, recipients were treated with a lymphodepleting dose of cyclophosphamide to enhance T-cell engraftment. In tumor therapy experiments, CAR19 or sCAR T-cells were adoptively transferred into huCD19Tg/0 mice bearing a syngeneic B-cell lymphoma engineered to express huCD19. To regulate sCAR T cell function, a switch protein was generated that contained the sCAR-specific PNE genetically fused to an anti-huCD19 Fab fragment. Recipients of sCAR T-cells were injected with the switch to link sCAR effector with huCD19+ target cells. Mice were monitored for survival, tumor burden (where appropriate), morbidity (as measured by weight loss and clinical scores), and peripheral blood lymphocyte frequency.CAR19 and sCAR T-cells functioned comparably regarding in vivo expansion and B-cell depletion. However, sCAR T-cells were better tolerated as evidenced by the recipients' enhanced survival, reduced weight loss, and improved clinical scores. Discontinuing switch administration allowed healthy B-cell frequencies to return to pretreatment levels.In our mouse model, sCAR T-cells killed huCD19+ healthy and malignant B-cells and were better tolerated than CAR19 cells. Our data suggest sCAR might be clinically superior to the current FDA-approved therapies for B-cell lymphomas due to the reduced acute and chronic morbidities and mortality, lower incidence and severity of side effects, and B-cell reconstitution on cessation of switch administration.

Human CD19-Targeted Mouse T Cells Induce B Cell Aplasia and Toxicity in Human CD19 Transgenic Mice.

The clinical success of chimeric antigen receptor (CAR) T cell therapy for CD19+ B cell malignancies can be limited by acute toxicities and immunoglobulin replacement needs due to B cell aplasia from persistent CAR T cells. Life-threatening complications include cytokine release syndrome and neurologic adverse events, the exact etiologies of which are unclear. To elucidate the underlying toxicity mechanisms and test potentially safer CAR T cells, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T cells were adoptively transferred into mice whose normal B cells express a hCD19 transgene at hemizygous levels. Compared to homozygous hCD19 transgenic mice that have ∼75% fewer circulating B cells, hemizygous mice had hCD19 frequencies and antigen density more closely simulating human B cells. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR T cells had undetectable tumor levels. Recipients experienced B cell aplasia and antigen- and dose-dependent acute toxicities mirroring patient complications. Interleukin-6 (IL-6), interferon γ (IFN-γ), and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 (and IFN-γ) blunted toxicity. Apparent behavioral abnormalities associated with decreased microglial cells point to CAR-T-cell-induced neurotoxicity. This model will prove useful in testing strategies designed to improve hCD19-specific CAR T cell safety.

Inductive modulation of tris(phosphinomethyl)phenylborate donation at group VI metals via borate phenyl substituent modification.

A series of zerovalent group VI metal complexes of tris(diisopropylphosphinomethyl)phenylborate ([PhB(CH2PiPr2)3]-, PhBPiPr3), including [PPN][M(CO)3(PhBPiPr3)] (M = Cr, Mo, W) and the first bimetallics in which PhBPiPr3 serves as a bridging ligand via binding M(CO)3 units at the three phosphorus atoms and the borate phenyl substituent, have been synthesized and fully characterized. Two new tris(phosphinomethyl)borates featuring 3,5-dimethylphenyl and 3,5-bis(trifluoromethyl)phenyl borate substituents were prepared as crystallographically characterized thallium salts, and metallated giving their inaugural transition metal complexes [PPN][M(CO)3(((3,5-Me)C6H3)BPPh3)] and [PPN][M(CO)3(((3,5-CF3)C6H3)BPPh3)]. A comparative ν(CO) infrared spectroscopic analysis and examination of half wave potentials assessed by cyclic voltammetry supports a ligand donor ranking of Tp > PhBPiPr3 ≥ Cp > PhBPPh3 > triphos. For these anionic complexes, in which a lower electrostatic contribution to zerovalent metal-PhBPR3 binding is likely operative relative to that present in the zwitterionic complexes most commonly prepared with tris(phosphinomethyl)borates, PhBPR3 ligands do not function as strongly donating scorpionates. Nevertheless, PhBPPh3 is a substantially stronger donor than triphos towards zerovalent M(CO)3; the half wave potentials of [Et4N][M(CO)3(PhBPPh3)] are ∼340 mV lower than those of M(CO)3(triphos). The potentials of the ((3,5-Me)C6H3)BPPh3 group VI metal tricarbonyl anions are more negative than those of the corresponding ((3,5-CF3)C6H3)BPPh3 group VI metal tricarbonyl anions by ∼50 mV, suggesting a modest, yet rational, tuning of PhBPPh3 donation via inductive modulation of the borate anion charge.