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). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

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. To address these limitations, we integrated RQR8, a multi-epitope molecule harboring a CD34 epitope and two CD20 mimotopes, alongside a CD19-targeting CAR, into the CD52 locus. Using CRISPR-Cas9 and adeno-associated virus-based donor vectors, some 60% of genome-edited T cells were CAR+/CD20+/CD34+/CD52- without further selection. This could be increased to >95% purity after CD34 tag-based positive selection. These epitope-switched CAR T cells retained cell killing competence against CD19+ tumor cells, and were resistant to alemtuzumab (anti-CD52) but sensitive to rituximab (anti-CD20) in complement-dependent cytotoxicity assays. In conclusion, gene editing-based multiple epitope switching represents a promising development with the potential to improve both the manufacturing procedure as well as the clinical safety of CAR T cells.

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. Near to 100% efficiency is reached using only 2.5 × 10-4 pmol of siRNA, and 1 × 10-3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo.

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. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.

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. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15-30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation.

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. To overcome this challenge, we established two simple cell-based CRS in vitro models that entail the co-culturing of leukemic B cells with CD19-targeting CAR T cells and primary monocytes from the same donor. Upon antigen encounter, CAR T cells upregulated CD40L and released GM-CSF which in turn stimulated the monocytes to secrete IL-6. To endorse these models, we demonstrated that neutralizing antibodies or genetic disruption of the CD40L and/or CSF2 loci in CAR T cells using CRISPR-Cas technology significantly reduced IL-6 secretion by bystander monocytes without affecting the cytolytic activity of the engineered lymphocytes in vitro. Overall, our cell-based models were able to recapitulate CRS in vitro, allowing us to validate mitigation strategies based on antibodies or genome editing.

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. Here, we developed a CRISPR-Cas9-based gene editing protocol that allowed us to knockout about 80% of the NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus in primary NK cells. In-depth phenotypic analysis confirmed significant reduction in NKG2A protein expression. Importantly, the KLRC1-edited NK cells showed significantly increased cytotoxicity against primary MM cells isolated from a small cohort of patients, and maintained the NK cell-specific cytokine production. In conclusion, KLRC1-editing in primary NK cells has the prospect of overcoming immune checkpoint inhibition in clinical applications.

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. Furthermore, CAR T cell therapy has the potential to be combined with other treatment modalities, such as androgen deprivation therapy, radiotherapy or chemotherapy, and could be applied as focal CAR T cell therapy for prostate cancer.

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. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a TRAC locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding TRAC locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/ß-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/ß-free CAR19 T cells at clinical scale.

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. In vitro 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. In vivo, focal injections of the PSMA CAR T cells eradicated established human prostate cancer xenografts in a preclinical mouse model. Moreover, systemic intravenous CAR T cell application significantly inhibited tumor growth in combination with non-ablative low-dose docetaxel chemotherapy, while docetaxel or CAR T cell application alone was not effective. In conclusion, the focal application of D7-derived CAR T cells and their combination with chemotherapy represent promising immunotherapeutic avenues to treat local and advanced prostate cancer in the clinic.

Modeling MyD88 Deficiency In Vitro 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 in vitro. To this end, we determined the immunophenotype of monocytes and macrophages derived from MyD88- and IRAK4-deficient patients. We recognized that macrophages derived from both patients were particularly poorly activated by streptococci, indicating that both signaling intermediates are essential for the immune response to facultative pathogens. To characterize this defect in more detail, we generated induced pluripotent stem cells (iPSCs) of fibroblasts derived from an MyD88-deficient patient. The underlying genetic defect was corrected using Sleeping Beauty transposon vectors encoding either the long (L) or the short (S) MYD88 isoform, respectively. Macrophages derived from these iPSC lines (iMacs) expressed typical macrophage markers, stably produced either MyD88 isoform, and showed robust phagocytic activity. Notably, iMacs expressing MyD88-L, but not MyD88-S, exhibited similar responses to external stimuli, including cytokine release patterns, as compared to genetically normal iMacs. Thus, the two MyD88 isoforms assume distinct functions in signaling. In conclusion, iPSC technology, in combination with efficient myeloid differentiation protocols, provides a valuable and inexhaustible source of macrophages, which can be used for disease modeling. Moreover, iPSC-derived macrophages may eventually aid in stabilizing MyD88-deficient patients during pyogenic infections.

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. This review summarizes the fundamentals of genome editing and the current state of research. It particularly focuses on the advances made in employing engineered nucleases in hematopoietic stem cells for the treatment of primary immunodeficiencies and hemoglobinopathies, provides a perspective of combining gene editing with the chimeric antigen receptor T cell technology, and concludes by presenting targeted epigenome editing as a novel potential treatment option.

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. To model X-SCID in vitro, we generated a mouse embryonic stem cell (ESC) line in which a disease-causing human IL2RG gene variant replaces the endogenous Il2rg locus. We developed a stage-specific T cell differentiation protocol to validate genetic correction of the common G691A mutation with transcription activator-like effector nucleases. While all ESC clones could be differentiated to hematopoietic precursor cells, stage-specific analysis of T cell maturation confirmed early arrest of T cell differentiation at the T cell progenitor stage in X-SCID cells. In contrast, genetically corrected ESCs differentiated to CD4 + or CD8 + single-positive T cells, confirming correction of the cellular X-SCID phenotype. This study emphasises the value of PSCs for disease modelling and underlines the significance of in vitro models as tools to validate genome editing strategies before clinical application.

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. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse 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. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.