CRISPR/Cas9 editing of NKG2A improves the efficacy of primary CD33-directed chimeric antigen receptor natural killer cells.

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells show antileukemic activity against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by the interaction between human leukocyte antigen (HLA)-E and the inhibitory receptor, NKG2A. Here, we describe a strategy that overcomes CAR-NK cell inhibition mediated by the HLA-E-NKG2A immune checkpoint.

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs).

Cell-Based Models of 'Cytokine Release Syndrome' Endorse CD40L and Granulocyte-Macrophage Colony-Stimulating Factor Knockout in Chimeric Antigen Receptor T Cells as Mitigation Strategy.

While chimeric antigen receptor (CAR) T cell therapy has shown promising outcomes among patients with hematologic malignancies, it has also been associated with undesirable side-effects such as cytokine release syndrome (CRS). CRS is triggered by CAR T-cell-based activation of monocytes, which are stimulated via the CD40L-CD40R axis or via uptake of GM-CSF to secrete proinflammatory cytokines. Mouse models have been used to model CRS, but working with them is labor-intensive and they are not amenable to screening approaches.

CRISPR-Cas9 based gene editing of the immune checkpoint NKG2A enhances NK cell mediated cytotoxicity against multiple myeloma.

Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity, and the possibility to be applied as 'off-the-shelf' product makes them highly attractive for cell-based immunotherapies. In patients with multiple myeloma (MM), an elevated number of NK cells has been correlated with higher overall-survival rate. However, NK cell function can be impaired by upregulation of inhibitory receptors, such as the immune checkpoint NKG2A.

The potential of CAR T cell therapy for prostate cancer.

Chimeric antigen receptor (CAR) T cell immunotherapy involves the genetic modification of the patient's own T cells so that they specifically recognize and destroy tumour cells. Considerable clinical success has been achieved using this technique in patients with lymphoid malignancies, but clinical studies that investigated treating solid tumours using this emerging technology have been disappointing. A number of developments might be able to increase the efficacy of CAR T cell therapy for treatment of prostate cancer, including improved trafficking to the tumour, techniques to overcome the immunosuppressive tumour microenvironment, as well as methods to enhance CAR T cell persistence, specificity and safety.

Automated generation of gene-edited CAR T cells at clinical scale.

The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit.

Targeted multi-epitope switching enables straightforward positive/negative selection of CAR T cells.

Chimeric antigen receptor (CAR) T cell technology has enabled successfully novel concepts to treat cancer patients, with substantial remission rates in lymphoid malignancies. This cell therapy is based on autologous T lymphocytes that are genetically modified to express a CAR that recognizes tumor-associated antigens and mediates the elimination of the respective tumor cells. Current limitations include laborious manufacturing procedures as well as severe immunological side effects upon administration of CAR T cells.

Modeling MyD88 Deficiency Provides New Insights in Its Function.

Inherited defects in MyD88 and IRAK4, two regulators in Toll-like receptor (TLR) signaling, are clinically highly relevant, but still incompletely understood. MyD88- and IRAK4-deficient patients are exceedingly susceptible to a narrow spectrum of pathogens, with ∼50% lethality in the first years of life. To better understand the underlying molecular and cellular characteristics that determine disease progression, we aimed at modeling the cellular response to pathogens .

PSMA-Directed CAR T Cells Combined with Low-Dose Docetaxel Treatment Induce Tumor Regression in a Prostate Cancer Xenograft Model.

While chimeric antigen receptor (CAR) T cell immunotherapy targeting CD19 has shown remarkable success in patients with lymphoid malignancies, the potency of CAR T cells in solid tumors is low so far. To improve the efficacy of CAR T cells targeting prostate carcinoma, we designed a novel CAR that recognizes a new epitope in the prostate-specific membrane antigen (PSMA) and established novel paradigms to apply CAR T cells in a preclinical prostate cancer model. characterization of the D7 single-chain antibody fragment-derived anti-PSMA CAR confirmed that the choice of the co-stimulatory domain is a major determinant of CAR T cell activation, differentiation, and exhaustion.

Biodegradable Nanocarriers Resembling Extracellular Vesicles Deliver Genetic Material with the Highest Efficiency to Various Cell Types.

Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co-transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells.

Targeted genome editing restores T cell differentiation in a humanized X-SCID pluripotent stem cell disease model.

The generation of T cells from pluripotent stem cells (PSCs) is attractive for investigating T cell development and validating genome editing strategies in vitro. X-linked severe combined immunodeficiency (X-SCID) is an immune disorder caused by mutations in the IL2RG gene and characterised by the absence of T and NK cells in patients. IL2RG encodes the common gamma chain, which is part of several interleukin receptors, including IL-2 and IL-7 receptors.

Genome and Epigenome Editing to Treat Disorders of the Hematopoietic System.

The possibility of editing complex genomes in a targeted fashion has revolutionized basic research as well as biomedical and biotechnological applications in the last 5 years. The targeted introduction of genetic changes has allowed researchers to create smart model systems for basic research, bio-engineers to modify crops and farm animals, and translational scientists to develop novel treatment approaches for inherited and acquired disorders for which curative treatment options are not yet available. With the rapid development of genome editing tools, in particular zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system, a wide range of therapeutic options have been-and will be-developed at an unprecedented speed, which will change the clinical routine of various disciplines in a revolutionary way.

Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model.

In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity.

TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity.

Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNA-guided endonucleases has been assessed to some extent, little data are available for transcription activator-like effector-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity.

Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery.

Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells.

TALE-PvuII fusion proteins--novel tools for gene targeting.

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module.