CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca2+ sensor GCaMP6S to visualize in real-time chemokine-induced Ca2+ signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo

DNA methylation changes during long-term in vitro cell culture are caused by epigenetic drift

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift

Efficient Genetic Safety Switches for Future Application of iPSC-Derived Cell Transplants

Induced pluripotent stem cell (iPSC)-derived cell products hold great promise as a potential cell source in personalized medicine. As concerns about the potential risk of graft-related severe adverse events, such as tumor formation from residual pluripotent cells, currently restrict their applicability, we established an optimized tool for therapeutic intervention that allows drug-controlled, specific and selective ablation of either iPSCs or the whole graft through genetic safety switches. To identify the best working system, different tools for genetic iPSC modification, promoters to express safety switches and different safety switches were combined. Suicide effects were slightly stronger when the suicide gene was delivered through lentiviral (LV) vectors compared to integration into the AAVS1 locus through TALEN technology. An optimized HSV-thymidine kinase and the inducible Caspase 9 both mediated drug-induced, efficient in vitro elimination of transgene-positive iPSCs. Choice of promoter allowed selective elimination of distinct populations within the graft: the hOct4 short response element restricted transgene expression to iPSCs, while the CAGs promoter ubiquitously drove expression in iPSCs and their progeny. Remarkably, both safety switches were able to prevent in vivo teratoma development and even effectively eliminated established teratomas formed by LV CAGs-transgenic iPSCs. These optimized tools to increase safety provide an important step towards clinical application of iPSC-derived transplants

Targeted Repair of p47-CGD Restores the Ability of iPSC-Derived Macrophages to Kill Bacteria

Chronic granulomatous disease (CGD) is caused by mutations in the NADPH oxidase and is characterized by defective phagocytes. The NAPDH oxidase is important for the production of reactive oxygen species that further regulate the phagosome milieu, such as the release of granules containing antimicrobial proteins, and the killing of bacteria inside the phagosome, which is defective in CGD patients. Only allogenic hematopoietic stem cell transplantation (HSCT) and retroviral gene therapy are curative treatment options for CGD patients. The first gene therapy trials for CGD were complicated by insertional mutagenesis and transgene silencing of the applied LTRdriven gamma-retroviral vector. More promising results were shown by recent studies with lentiviral SIN vectors and a myeloid-specific promoter. However, targeted gene correction using CRISPR-Cas9 would avoid the risk of vector-mediated insertional mutagenesis and silencing of the therapeutic gene. In our study, we developed a gene editing approach to correct p47phox-deficiency, which affects about25% of CGD patients. Over 90% of these patients carry the mutation c.75 76delGT (ΔGT) in exon 2 of NCF1 (encodes p47phox). This highprevalence most likely originates via gene conversion from one of the two pseudogenes NCF1B/C, which are over 99% homologous to NCF1 and carry the aforementioned ΔGT mutation. For proof-of-principle, we corrected p47phox-deficient induced pluripotent stem cells (iPSC)by inserting a minigene donor construct into intron 1 of NCF1. We chose this position, which differs by three additional nucleotides from the pseudogenes, to avoid cleavage in the pseudogenes and, thus, potential chromosomal instability due to multiple DNA doublestrand breaks. To analyze functionality, genetically corrected clones were differentiated into granulocytes and macrophages. Corrected granulocytes expressed p47phoxsimilar to wild type levels, displayedNADPH oxidase activity and were able to form neutrophil extracellular traps, which enable granulocytes to kill microbes extracellularly. Finally, iPSC-derived macrophages were infected with GFP-labeled E. coli. After phagocytosis, corrected macrophages significantly reduced the amount of living bacteria inside their phagosomes as measured by colony-forming units in plated cell lysates compared to uncorrected macrophages. In summary, we demonstrated that the specific insertion of a minigene into intron 1 of NCF1 corrects p47phox-deficiency.Moreover, the pseudogenes remained intact with our strategy, which could be of importance as the pseudogenes might have functional roles. We showed that the corrected iPSC-derived granulocytes and macrophages have all the features necessary to fight infections. In the past, some patients benefited from allogenic granulocyte transfusions. However, due to alloimmunization, this therapy is contraindicative for subsequent HSCT. We propose that autologous iPSC-derived granulocytes and macrophages could be applied therapeutically in the future to combat refractory infections prior to HSCT

Human Teratoma-Derived Hematopoiesis Is a Highly Polyclonal Process Supported by Human Umbilical Vein Endothelial Cells

Summary: Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model. : Schambach and colleagues show evidence that hematopoiesis in hiPSC-derived teratomas occurs by endothelial-to-hematopoietic transition in a highly polyclonal manner. This process was supported by the presence of HUVECs in the NSGS mouse, which provides human SCF, IL-3, and GM-CSF. The established hematopoiesis model can potentially be used for disease modeling and improvement of hematopoietic differentiation protocols. Keywords: hematopoiesis, teratoma, hiPSC, EHT, genetic barcoding, HUVECs, embryogenesi

Targeted Repair of p47-CGD in iPSCs by CRISPR/Cas9: Functional Correction without Cleavage in the Highly Homologous Pseudogenes

Mutations in the NADPH oxidase, which is crucial for the respiratory burst in phagocytes, result in chronic granulomatous disease (CGD). The only curative treatment option for CGD patients, who suffer from severe infections, is allogeneic bone marrow transplantation. Over 90% of patients with mutations in the p47phox subunit of the oxidase complex carry the deletion c.75 76delGT (ΔGT). This frequent mutation most likely originates via gene conversion from one of the two pseudogenes NCF1B or NCF1C, which are highly homologous to NCF1 (encodes p47phox) but carry the ΔGT mutation. We applied CRISPR/Cas9 to generate patient-like p47-ΔGT iPSCs for disease modeling. To avoid unpredictable chromosomal rearrangements by CRISPR/Cas9-mediated cleavage in the pseudogenes, we developed a gene-correction approach to specifically target NCF1 but leave the pseudogenes intact. Functional assays revealed restored NADPH oxidase activity and killing of bacteria in corrected phagocytes as well as the specificity of this approach

Herpes simplex virus type 1 modifies the protein composition of extracellular vesicles to promote neurite outgrowth and neuroinfection

ABSTRACTThe highly prevalent herpes simplex virus type 1 (HSV-1) causes a range of diseases, including cold sores, blinding keratitis, and life-threatening encephalitis. HSV-1 initially replicates in epithelial cells, enters the peripheral nervous system via neurites, and establishes lifelong infection in the neuronal cell bodies. Neurites are highly dynamic structures that grow or retract in response to attractive or repulsive cues, respectively. Here, we show that infection with HSV-1, but not with a mutant virus lacking glycoprotein G (gG), reduced the repulsive effect of epithelial cells on neurite outgrowth and facilitated HSV-1 invasion of neurons. HSV-1 gG was required and sufficient to induce neurite outgrowth by modifying the protein composition of extracellular vesicles, increasing the amount of neurotrophic and neuroprotective proteins, including galectin-1. Antibodies directed against galectin-1 neutralized the capacity of extracellular vesicles released from HSV-1-infected cells to promote neurite outgrowth. Our study provides new insights into the neurotropism of HSV-1 and identifies a viral protein that modifies the protein composition of extracellular vesicles to stimulate neurite outgrowth and invasion of the nervous system.IMPORTANCEHerpes simplex virus type 1 (HSV-1) must infect neurites (or nerve endings) to establish a chronic infection in neurons. Neurites are highly dynamic structures that retract or grow in the presence of repulsive or attractive proteins. Some of these proteins are released by epithelial cells in extracellular vesicles and act upon interaction with their receptor present on neurites. We show here that HSV-1 infection of epithelial cells modulated their effect on neurites, increasing neurite growth. Mechanistically, HSV-1 glycoprotein G (gG) modifies the protein composition of extracellular vesicles released by epithelial cells, increasing the amount of attractive proteins that enhance neurite outgrowth and facilitate neuronal infection. These results could inform of therapeutic strategies to block HSV-1 induction of neurite outgrowth and, thereby, neuronal infection

Predicting genotoxicity of viral vectors for stem cell gene therapy using gene expression-based machine learning

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials

Predicting genotoxicity of viral vectors for stem cell gene therapy using gene expression-based machine learning

status: submitte