Epitope editing enables targeted immunotherapy of acute myeloid leukaemia.

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia1,2, the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens3-5. Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.

A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML.

HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.

Lpr-induced systemic autoimmunity is unaffected by mast cell deficiency.

The function of mast cells in allergic and organ-specific autoimmune responses is highly controversial. In the current study, we aimed to dissect the role of mast cells in systemic autoimmunity in the B6(lpr/lpr) mouse, a spontaneous model of systemic lupus erythematosus. B6(lpr/lpr) mice were interbred with C57Bl/6-Kit(W-sh/W-sh) (Wsh) mice, resulting in mast cell deficiency. The offspring from this cross (Lpr/Wsh mice) developed symptoms of lupus of the same severity as B6(lpr/lpr) mice. Loss of mast cells on the Lpr background did not alter autoantibody production, proteinuria, the composition of T and B cell populations or autoimmune pathology. Reduced c-Kit expression did drive expanded splenomegaly and impeded interleukin-4 production by CD4(+) cells, suggesting minor functions for mast cells. In general, we conclude that mast cell deficiency and c-Kit deficiency do not play a role in the pathogenesis of lupus in B6(lpr/lpr) mice.

Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells.

DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus- and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of non-retroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous TP53/Trp53 genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34+ stem and progenitor cells. Strikingly, co-packaging of Cas9 mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.

Airway basal cells show a dedifferentiated KRT17highPhenotype and promote fibrosis in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study, we focus on the properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing (scRNAseq) of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17high PTENlow dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2-/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNAseq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrate that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model.

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.

Competitive sgRNA Screen Identifies p38 MAPK as a Druggable Target to Improve HSPC Engraftment.

Previous gene therapy trials for X-linked chronic granulomatous disease (X-CGD) lacked long-term engraftment of corrected hematopoietic stem and progenitor cells (HSPCs). Chronic inflammation and high levels of interleukin-1 beta (IL1B) might have caused aberrant cell cycling in X-CGD HSPCs with a concurrent loss of their long-term repopulating potential. Thus, we performed a targeted CRISPR-Cas9-based sgRNA screen to identify candidate genes that counteract the decreased repopulating capacity of HSPCs during gene therapy. The candidates were validated in a competitive transplantation assay and tested in a disease context using IL1B-challenged or X-CGD HSPCs. The sgRNA screen identified Mapk14 (p38) as a potential target to increase HSPC engraftment. Knockout of p38 prior to transplantation was sufficient to induce a selective advantage. Inhibition of p38 increased expression of the HSC homing factor CXCR4 and reduced apoptosis and proliferation in HSPCs. For potential clinical translation, treatment of IL1B-challenged or X-CGD HSPCs with a p38 inhibitor led to a 1.5-fold increase of donor cell engraftment. In summary, our findings demonstrate that p38 may serve as a potential druggable target to restore engraftment of HSPCs in the context of X-CGD gene therapy.

CXCR4/MIF axis amplifies tumor growth and epithelial-mesenchymal interaction in non-small cell lung cancer.

Overexpression of C-X-C chemokine receptor type 4 (CXCR4) has been shown in several cancers, including non-small cell lung cancer (NSCLC) and is linked to early metastasis and worse prognosis. The crosstalk between cancer cells and tumor stroma promotes the growth and metastasis and CXCR4 signaling is a key element of this crosstalk. To test the effects of CXCR4 overexpression (CXCR4-OE), we transduced the human NSCLC cell line A549 by using a lentiviral vector. A 3D cell culture model showed generations of tumorspheres and the effects derived by the co-culturing of lung fibroblasts. Using a xenograft mouse model, we also studied the effects of CXCR4-OE in pulmonary cell engraftment and tumor burden in vivo. Our data indicate that CXCR4-OE leads to increased tumorsphere formation and epithelial-mesenchymal transition (EMT). CXCR4-OE by A549 cells resulted in a significant increase in the production of the CXCR4-ligand macrophage migration inhibitory factor (MIF) compared to those transduced with an empty vector (EV) or in which the CXCR4 expression was deleted (KO). In our in vitro system, we did not detect any production of the canonical CXCR4 ligand CXCL12. Autocrine MIF production and CXCR4 signaling are part of a self-perpetuating loop that amplifies tumor growth and EMT. Co-culture with lung fibroblasts further increased tumorsphere formation, partially driven by an increase in IL-6 production. When A549 cells were injected into murine lungs, we observed more abundant and significantly larger tumor lesions in recipients of CXCR4-OE A549 cells compared to those receiving EV or KO cells, consistent with our in vitro findings. Treatment of mice with the MIF antagonist ISO-1 resulted in significantly less tumor burden. In conclusion, our data highlight the role of the CXCR4-OE/MIF/IL-6 axis in epithelial mesenchymal crosstalk and NSCLC progression.

Differential Transgene Silencing of Myeloid-Specific Promoters in the AAVS1 Safe Harbor Locus of Induced Pluripotent Stem Cell-Derived Myeloid Cells.

Targeted integration into a genomic safe harbor, such as the AAVS1 locus on chromosome 19, promises predictable transgene expression and reduces the risk of insertional mutagenesis in the host genome. The application of gamma-retroviral long terminal repeat (LTR)-driven vectors, which semirandomly integrate into the genome, has previously caused severe adverse events in some clinical studies due to transactivation of neighboring proto-oncogenes. Consequently, the site-specific integration of a therapeutic transgene into a genomic safe harbor locus would allow stable genetic correction with a reduced risk of insertional mutagenesis. However, recent studies revealed that transgene silencing, especially in case of weaker cell type-specific promoters, can occur in the AAVS1 locus of human pluripotent stem cells (PSCs) and can impede transgene expression during differentiation. In this study, we aimed to correct p47phox deficiency, which is the second most common cause of chronic granulomatous disease, by insertion of a therapeutic p47phox transgene into the AAVS1 locus of human induced PSCs (iPSCs) using CRISPR-Cas9. We analyzed transgene expression and functional correction from three different myeloid-specific promoters (miR223, CatG/cFes, and myeloid-related protein 8 [MRP8]). Upon myeloid differentiation of corrected iPSC clones, we observed that the miR223 and CatG/cFes promoters achieved therapeutically relevant levels of p47phox expression and nicotinamide adenine dinucleotide phosphate oxidase activity, whereas the MRP8 promoter was less efficient. Analysis of the different promoters revealed high CpG methylation of the MRP8 promoter in differentiated cells, which correlated with the transgene expression data. In summary, we identified the miR223 and CatG/cFes promoters as cell type-specific promoters that allow stable transgene expression in the AAVS1 locus of iPSC-derived myeloid cells. Our findings further indicate that promoter silencing can occur in the AAVS1 safe harbor locus in differentiated hematopoietic cells and that a comparison of different promoters is necessary to achieve optimal transgene expression for therapeutic application of iPSC-derived cells.

Preclinical Evaluation of a Novel Lentiviral Vector Driving Lineage-Specific BCL11A Knockdown for Sickle Cell Gene Therapy.

In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNAmiR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle ß-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34+ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.

Hematopoietic stem-cell senescence and myocardial repair - Coronary artery disease genotype/phenotype analysis of post-MI myocardial regeneration response induced by CABG/CD133+ bone marrow hematopoietic stem cell treatment in RCT PERFECT Phase 3.

BACKGROUND: Bone marrow stem cell clonal dysfunction by somatic mutation is suspected to affect post-infarction myocardial regeneration after coronary bypass surgery (CABG). METHODS: Transcriptome and variant expression analysis was studied in the phase 3 PERFECT trial post myocardial infarction CABG and CD133+ bone marrow derived hematopoetic stem cells showing difference in left ventricular ejection fraction (∆LVEF) myocardial regeneration Responders (n=14; ∆LVEF +16% day 180/0) and Non-responders (n=9; ∆LVEF -1.1% day 180/0). Subsequently, the findings have been validated in an independent patient cohort (n=14) as well as in two preclinical mouse models investigating SH2B3/LNK antisense or knockout deficient conditions. FINDINGS: 1. Clinical: R differed from NR in a total of 161 genes in differential expression (n=23, q<0•05) and 872 genes in coexpression analysis (n=23, q<0•05). Machine Learning clustering analysis revealed distinct RvsNR preoperative gene-expression signatures in peripheral blood acorrelated to SH2B3 (p<0.05). Mutation analysis revealed increased specific variants in RvsNR. (R: 48 genes; NR: 224 genes). 2. Preclinical:SH2B3/LNK-silenced hematopoietic stem cell (HSC) clones displayed significant overgrowth of myeloid and immune cells in bone marrow, peripheral blood, and tissue at day 160 after competitive bone-marrow transplantation into mice. SH2B3/LNK-/- mice demonstrated enhanced cardiac repair through augmenting the kinetics of bone marrow-derived endothelial progenitor cells, increased capillary density in ischemic myocardium, and reduced left ventricular fibrosis with preserved cardiac function. 3. VALIDATION: Evaluation analysis in 14 additional patients revealed 85% RvsNR (12/14 patients) prediction accuracy for the identified biomarker signature. INTERPRETATION: Myocardial repair is affected by HSC gene response and somatic mutation. Machine Learning can be utilized to identify and predict pathological HSC response. FUNDING: German Ministry of Research and Education (BMBF): Reference and Translation Center for Cardiac Stem Cell Therapy - FKZ0312138A and FKZ031L0106C, German Ministry of Research and Education (BMBF): Collaborative research center - DFG:SFB738 and Center of Excellence - DFG:EC-REBIRTH), European Social Fonds: ESF/IV-WM-B34-0011/08, ESF/IV-WM-B34-0030/10, and Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany. Japanese Ministry of Health : Health and Labour Sciences Research Grant (H14-trans-001, H17-trans-002) TRIAL REGISTRATION: ClinicalTrials.gov NCT00950274.

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.

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

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.

Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype.

Reducing expression of the fetal hemoglobin (HbF) repressor BCL11A leads to a simultaneous increase in γ-globin expression and reduction in ß-globin expression. Thus, there is interest in targeting BCL11A as a treatment for ß-hemoglobinopathies, including sickle cell disease (SCD) and ß-thalassemia. Here, we found that using optimized shRNAs embedded within an miRNA (shRNAmiR) architecture to achieve ubiquitous knockdown of BCL11A profoundly impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs) despite a reduction in nonspecific cellular toxicities. BCL11A knockdown was associated with a substantial increase in S/G2-phase human HSCs after engraftment into immunodeficient (NSG) mice, a phenotype that is associated with HSC exhaustion. Lineage-specific, shRNAmiR-mediated suppression of BCL11A in erythroid cells led to stable long-term engraftment of gene-modified cells. Transduced primary normal or SCD human HSCs expressing the lineage-specific BCL11A shRNAmiR gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60%-70% γ-chain expression (vs. < 10% for negative control) and a corresponding increase in HbF. Transplantation of gene-modified murine HSCs from Berkeley sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key pathophysiological biomarkers of SCD. These data form the basis for a clinical trial application for treating sickle cell disease.