CD45-Directed CAR-T Cells with CD45 Knockout Efficiently Kill Myeloid Leukemia and Lymphoma Cells In Vitro Even after Extended Culture.

BACKGROUND: CAR-T cell therapy has shown impressive results and is now part of standard-of-care treatment of B-lineage malignancies, whereas the treatment of myeloid diseases has been limited by the lack of suitable targets. CD45 is expressed on almost all types of blood cells including myeloid leukemia cells, but not on non-hematopoietic tissue, making it a potential target for CAR-directed therapy. Because of its high expression on T and NK cells, fratricide is expected to hinder CD45CAR-mediated therapy. Due to its important roles in effector cell activation, signal transduction and cytotoxicity, CD45 knockout aimed at preventing fratricide in T and NK cells has been expected to lead to considerable functional impairment. METHODS: CD45 knockout was established on T and NK cell lines using CRISPR/Cas9-RNPs and electroporation, and the successful protocol was transferred to primary T cells. A combined protocol was developed enabling CD45 knockout and retroviral transduction with a third-generation CAR targeting CD45 or CD19. The functionality of CD45ko effector cells, CD45ko/CD45CAR-T and CD45ko/CD19CAR-T cells was studied using proliferation as well as short- and long-term cytotoxicity assays. RESULTS: As expected, the introduction of a CD45-CAR into T cells resulted in potent fratricide that can be avoided by CD45 knockout. Unexpectedly, the latter had no negative impact on T- and NK-cell proliferation in vitro. Moreover, CD45ko/CD45CAR-T cells showed potent cytotoxicity against CD45-expressing AML and lymphoma cell lines in short-term and long-term co-culture assays. A pronounced cytotoxicity of CD45ko/CD45CAR-T cells was maintained even after four weeks of culture. In a further setup, we confirmed the conserved functionality of CD45ko cells using a CD19-CAR. Again, the proliferation and cytotoxicity of CD45ko/CD19CAR-T cells showed no differences from those of their CD45-positive counterparts in vitro. CONCLUSIONS: We report the efficient production of highly and durably active CD45ko/CAR-T cells. CD45 knockout did not impair the functionality of CAR-T cells in vitro, irrespective of the target antigen. If their activity can be confirmed in vivo, CD45ko/CD45CAR-T cells might, for example, be useful as part of conditioning regimens prior to stem cell transplantation.

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/NKp44-interactions in PSC. DESIGN: Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. RESULTS: NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1*02:01~B1*01:01 (OR 1.99, p=6.7×10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1*02:01-DPB1*01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1*02:01-DPB1*01:01 after IFN-γ-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1*02:01-DPB1*01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1-KO organoids. CONCLUSION: Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01~DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1*02:01-DPB1*01:01 expressed on cholangiocytes in PSC pathogenesis.

Chaperone-mediated autophagy in neuronal dendrites utilizes activity-dependent lysosomal exocytosis for protein disposal.

The complex morphology of neurons poses a challenge for proteostasis because the majority of lysosomal degradation machinery is present in the cell soma. In recent years, however, mature lysosomes were identified in dendrites, and a fraction of those appear to fuse with the plasma membrane and release their content to the extracellular space. Here, we report that dendritic lysosomes are heterogeneous in their composition and that only those containing lysosome-associated membrane protein (LAMP) 2A and 2B fuse with the membrane and exhibit activity-dependent motility. Exocytotic lysosomes dock in close proximity to GluN2B-containing N-methyl-D-aspartate-receptors (NMDAR) via an association of LAMP2B to the membrane-associated guanylate kinase family member SAP102/Dlg3. NMDAR-activation decreases lysosome motility and promotes membrane fusion. We find that chaperone-mediated autophagy is a supplier of content that is released to the extracellular space via lysosome exocytosis. This mechanism enables local disposal of aggregation-prone proteins like TDP-43 and huntingtin.

Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs.

The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application.

CRISPR-to-Kill (C2K)-Employing the Bacterial Immune System to Kill Cancer Cells.

CRISPR/Cas9 was described as a bacterial immune system that uses targeted introduction of DNA double-strand breaks (DSBs) to destroy invaders. We hypothesized that we can analogously employ CRISPR/Cas9 nucleases to kill cancer cells by inducing maximal numbers of DSBs in their genome and thus triggering programmed cell death. To do so, we generated CRISPR-to-kill (C2K) lentiviral particles targeting highly repetitive Short Interspersed Nuclear Element-Alu sequences. Our Alu-specific sgRNA has more than 15,000 perfectly matched target sites within the human genome. C2K-Alu-vectors selectively killed human, but not murine cell lines. More importantly, they efficiently inhibited the growth of cancer cells including patient-derived glioblastoma cell lines resistant to high-dose irradiation. Our data provide proof-of-concept for the potential of C2K as a novel treatment strategy overcoming common resistance mechanisms. In combination with tumor-targeting approaches, the C2K system might therefore represent a promising tool for cancer gene therapy.

LATE-a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects.

Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.

Optimisation of a TALE nuclease targeting the HIV co-receptor CCR5 for clinical application.

Disruption of the C-C-Chemokine-receptor-5 (CCR5) gene induces resistance towards CCR5-tropic HIV. Here we optimised our previously described CCR5-Uco-TALEN and its delivery by mRNA electroporation. The novel variant, CCR5-Uco-hetTALEN features an obligatory heterodimeric Fok1-cleavage domain, which resulted in complete abrogation of off-target activity at previously found homodimeric as well as 7/8 in silico predicted, potential heterodimeric off-target sites, the only exception being highly homologous CCR2. Prevailing 18- and 10-bp deletions at the on-target site revealed microhomology-mediated end-joining as a major repair pathway. Notably, the CCR5Δ55-60 protein resulting from the 18-bp deletion was almost completely retained in the cytosol. Simultaneous cutting at CCR5 and CCR2 induced rearrangements, mainly 15-kb deletions between the cut sites, in up to 2% of T cells underlining the necessity to restrict TALEN expression. We optimised in vitro mRNA production and showed that CCR5-on- and CCR2 off-target activities of CCR5-Uco-hetTALEN were limited to the first 72 and 24-48 h post-mRNA electroporation, respectively. Using single-cell HRMCA, we discovered high rates of TALEN-induced biallelic gene editing of CCR5, which translated in large numbers of CCR5-negative cells resistant to HIVenv-pseudotyped lentiviral vectors. We conclude that CCR5-Uco-hetTALEN transfected by mRNA electroporation facilitates specific, high-efficiency CCR5 gene-editing (30%-56%) and it is highly suited for clinical translation subject to further characterisation of off-target effects.

Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition.

The redesign of a macromolecular binding interface and corresponding alteration of recognition specificity is a challenging endeavor that remains recalcitrant to computational approaches. This is particularly true for the redesign of DNA binding specificity, which is highly dependent upon bending, hydrogen bonds, electrostatic contacts, and the presence of solvent and counterions throughout the molecular interface. Thus, redesign of protein-DNA binding specificity generally requires iterative rounds of amino acid randomization coupled to selections. Here, we describe the importance of scaffold thermostability for protein engineering, coupled with a strategy that exploits the protein's specificity profile, to redesign the specificity of a pair of meganucleases toward three separate genomic targets. We determine and describe a series of changes in protein sequence, stability, structure, and activity that accumulate during the engineering process, culminating in fully retargeted endonucleases.

Identification of protein structural elements responsible for the diversity of sequence preferences among Mini-III RNases.

Many known endoribonucleases select their substrates based on the presence of one or a few specific nucleotides at or near the cleavage site. In some cases, selectivity is also determined by the structural features of the substrate. We recently described the sequence-specific cleavage of double-stranded RNA by Mini-III RNase from Bacillus subtilis in vitro. Here, we characterized the sequence specificity of eight other members of the Mini-III RNase family from different bacterial species. High-throughput analysis of the cleavage products of Φ6 bacteriophage dsRNA indicated subtle differences in sequence preference between these RNases, which were confirmed and characterized by systematic analysis of the cleavage kinetics of a set of short dsRNA substrates. We also showed that the sequence specificities of Mini-III RNases are not reflected by different binding affinities for cognate and non-cognate sequences, suggesting that target selection occurs predominantly at the cleavage step. We were able to identify two structural elements, the α4 helix and α5b-α6 loop that were involved in target selection. Characterization of the sequence specificity of the eight Mini-III RNases may provide a basis for better understanding RNA substrate recognition by Mini-III RNases and adopting these enzymes and their engineered derivatives as tools for RNA research.

Sequence-specific endoribonucleases.

Ribonucleases are nucleolytic enzymes that commonly occur in living organisms and act by cleaving RNA molecules. These enzymes are involved in basic cellular processes, including the RNA maturation that accompanies the formation of functional RNAs, as well as RNA degradation that enables removal of defective or dangerous molecules or ones that have already fulfilled their cellular functions. RNA degradation is also one of the main processes that determine the amount of transcripts in the cell and thus it makes an important element of the gene expression regulation system. Ribonucleases can catalyse reactions involving RNA molecules containing specific sequences, structures or sequences within a specific structure, they can also cut RNAs non-specifically. In this article, we discuss ribonucleases cleaving the phosphodiester bond inside RNA molecules within or close to particular sequences. We also present examples of protein engineering of ribonucleases towards the development of molecular tools for sequence-specific cleavage of RNA.

Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

Cysteine-rich receptor-like kinase CRK5 as a regulator of growth, development, and ultraviolet radiation responses in Arabidopsis thaliana.

In plants, receptor-like protein kinases play essential roles in signal transduction by recognizing extracellular stimuli and activating the downstream signalling pathways. Cysteine-rich receptor-like kinases (CRKs) constitute a large subfamily of receptor-like protein kinases, with 44 members in Arabidopsis thaliana. They are distinguished by the novel C-X8-C-X2-C motif (DUF26) in the extracellular domains. One of them, CRK5, is an important component of the biochemical machinery involved in the regulation of essential physiological processes. Functional characterization of crk5 mutant plants showed their clear phenotype, manifested by impaired stomatal conductance and accelerated senescence. This phenotype correlated with accumulation of reactive oxygen species, higher foliar levels of ethylene and salicylic acid, and increased transcript abundance for genes associated with signalling pathways corresponding to these hormones. Moreover, the crk5 plants displayed enhanced cell death and oxidative damage in response to ultraviolet radiation. Complementation of CRK5 mutation managed to recover the wild-type phenotype, indicating an essential role of this gene in the regulation of growth, development, and acclimatory responses.

Sequence-specific cleavage of dsRNA by Mini-III RNase.

Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA.

Tobacco LSU-like protein couples sulphur-deficiency response with ethylene signalling pathway.

Most genes from the plant-specific family encoding Response to Low Sulphur (LSU)-like proteins are strongly induced in sulphur (S)-deficient conditions. The exact role of these proteins remains unclear; however, some data suggest their importance for plants' adjustment to nutrient deficiency and other environmental stresses. This work established that the regulation of ethylene signalling is a part of plants' response to S deficiency and showed the interaction between UP9C, a tobacco LSU family member, and one of the tobacco isoforms of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO2A). Increase in ethylene level induced by S deficiency does not take place in tobacco plants with UP9C expressed in an antisense orientation. Based on transcriptomics data, this work also demonstrated that the majority of tobacco's response to S deficiency is misregulated in plants expressing UP9C-antisense. A link between response to S deficiency, ethylene sensing, and LSU-like proteins was emphasized by changes in expression of the genes encoding ethylene receptors and F-box proteins specific for the ethylene pathway.