Hematopoietic Cell Autonomous Disruption of Hematopoiesis in a Germline Loss-of-function Mouse Model of RUNX1-FPD.

RUNX1 familial platelet disorder (RUNX1-FPD) is a hematopoietic disorder caused by germline loss-of-function mutations in the RUNX1 gene and characterized by thrombocytopathy, thrombocytopenia, and an increased risk of developing hematologic malignancies, mostly of myeloid origin. Disease pathophysiology has remained incompletely understood, in part because of a shortage of in vivo models recapitulating the germline RUNX1 loss of function found in humans, precluding the study of potential contributions of non-hematopoietic cells to disease pathogenesis. Here, we studied mice harboring a germline hypomorphic mutation of one Runx1 allele with a loss-of-function mutation in the other Runx1 allele (Runx1 L148A/- mice), which display many hematologic characteristics found in human RUNX1-FPD patients. Runx1 L148A/- mice displayed robust and pronounced thrombocytopenia and myeloid-biased hematopoiesis, associated with an HSC intrinsic reconstitution defect in lymphopoiesis and expansion of myeloid progenitor cell pools. We demonstrate that specific deletion of Runx1 from bone marrow stromal cells in Prrx1-cre;Runx1 fl/fl mice did not recapitulate these abnormalities, indicating that the hematopoietic abnormalities are intrinsic to the hematopoietic lineage, and arguing against a driving role of the bone marrow microenvironment. In conclusion, we report a RUNX1-FPD mouse model faithfully recapitulating key characteristics of human disease. Findings do not support a driving role of ancillary, non-hematopoietic cells in the disruption of hematopoiesis under homeostatic conditions.

A congenital CSF3R mutation in chronic neutropenia reveals a vital role for a cytokine receptor extracellular hinge motif in the response to granulocyte colony-stimulating factor.

We describe a patient with congenital neutropenia (CN) with a homozygous germline mutation in the colony-stimulating factor 3 receptor gene (CSF3R). The patient's bone marrow shows lagging neutrophil development with subtle left shift and unresponsiveness to CSF3 in in vitro colony assays. This patient illustrates that the di-proline hinge motif in the extracellular cytokine receptor homology domain of CSF3R is critical for adequate neutrophil production, but dispensable for in vivo terminal neutrophil maturation. This report underscores that CN patients with inherited CSF3R mutations should be marked as a separate clinical entity, characterized by a failure to respond to CSF3.

Validation and clinical application of transactivation assays for RUNX1 variant classification.

Familial platelet disorder with associated myeloid malignancies (RUNX1-familial platelet disorder [RUNX1-FPD]) is caused by heterozygous pathogenic germline variants of RUNX1. In the present study, we evaluate the applicability of transactivation assays to investigate RUNX1 variants in different regions of the protein. We studied 11 variants to independently validate transactivation assays supporting variant classification following the ClinGen Myeloid Malignancies Variant Curation Expert Panel guidelines. Variant classification is key for the translation of genetic findings. We showed that new assays need to be developed to assess C-terminal RUNX1 variants. Two variants of uncertain significance (VUS) were reclassified to likely pathogenic. Additionally, our analyses supported the (likely) pathogenic classification of 2 other variants. We demonstrated functionality of 4 VUS, but reclassification to (likely) benign was challenging and suggested the need for reevaluating current classification guidelines. Finally, clinical utility of our assays was illustrated in the context of 7 families. Our data confirmed RUNX1-FPD suspicion in 3 families with RUNX1-FPD-specific family history, whereas for 3 variants identified in RUNX1-FPD-nonspecific families, no functional defect was detected. Applying functional assays to support RUNX1 variant classification can be essential for adequate care of index patients and their relatives at risk. It facilitates translation of genetic data into personalized medicine.

Ready for Repair? Gene Editing Enters the Clinic for the Treatment of Human Disease.

We present an overview of clinical trials involving gene editing using clustered interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs) and discuss the underlying mechanisms. In cancer immunotherapy, gene editing is applied ex vivo in T cells, transgenic T cell receptor (tTCR)-T cells, or chimeric antigen receptor (CAR)-T cells to improve adoptive cell therapy for multiple cancer types. This involves knockouts of immune checkpoint regulators such as PD-1, components of the endogenous TCR and histocompatibility leukocyte antigen (HLA) complex to generate universal allogeneic CAR-T cells, and CD7 to prevent self-destruction in adoptive cell therapy. In cervix carcinoma caused by human papillomavirus (HPV), E6 and E7 genes are disrupted using topically applied gene editing machinery. In HIV infection, the CCR5 co-receptor is disrupted ex vivo to generate HIV-resistant T cells, CAR-T cells, or hematopoietic stem cells. In ß-thalassemia and sickle cell disease, hematopoietic stem cells are engineered ex vivo to induce the production of fetal hemoglobin. AAV-mediated in vivo gene editing is applied to exploit the liver for systemic production of therapeutic proteins in hemophilia and mucopolysaccharidoses, and in the eye to restore splicing of the CEP920 gene in Leber's congenital amaurosis. Close consideration of safety aspects and education of stakeholders will be essential for a successful implementation of gene editing technology in the clinic.

Sharpening the Molecular Scissors: Advances in Gene-Editing Technology.

The ability to precisely modify human genes has been made possible by the development of tools such as meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas. These now make it possible to generate targeted deletions, insertions, gene knock outs, and point variants; to modulate gene expression by targeting transcription factors or epigenetic machineries to DNA; or to target and modify RNA. Endogenous repair mechanisms are used to make the modifications required in DNA; they include non-homologous end joining, homology-directed repair, homology-independent targeted integration, microhomology-mediated end joining, base-excision repair, and mismatch repair. Off-target effects can be monitored using in silico prediction and sequencing and minimized using Cas proteins with higher accuracy, such as high-fidelity Cas9, enhanced-specificity Cas9, and hyperaccurate Cas9. Alternatives to Cas9 have been identified, including Cpf1, Cas12a, Cas12b, and smaller Cas9 orthologs such as CjCas9. Delivery of gene-editing components is performed ex vivo using standard techniques or in vivo using AAV, lipid nanoparticles, or cell-penetrating peptides. Clinical development of gene-editing technology is progressing in several fields, including immunotherapy in cancer treatment, antiviral therapy for HIV infection, and treatment of genetic disorders such as ß-thalassemia, sickle cell disease, lysosomal storage disorders, and retinal dystrophy. Here we review these technological advances and the challenges to their clinical implementation.