Eight induced pluripotent stem cell lines (iPSCs) derived from two patients with Leukocyte adhesion deficiency Type I (LAD I) with mutations in the ITGB2 gene.

Leukocyte Adhesion Deficiency Type I (LAD I) is a rare inborn error of immunity caused by mutations in the ITGB2 gene coding for β2-integrin CD18 on the surface of leukocytes. Affected patients display severe clinical manifestations with life threatening infections and inflammatory complications due to an impaired ability of leukocytes to transmigrate from the blood vessel to the tissue. Here we describe the generation of eight induced pluripotent stem cell lines from two patients with LAD I and mutations in the ITGB2 gene.

Targeted knock-in of cDNA into the locus leads to myeloid phenotypic correction of p47 -deficient chronic granulomatous disease.

p47 -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 () gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 expression under the control of the endogenous locus.

Expression of a large coding sequence: Gene therapy vectors for Ataxia Telangiectasia.

Ataxia telangiectasia is a monogenetic disorder caused by mutations in the ATM gene. Its encoded protein kinase ATM plays a fundamental role in DNA repair of double strand breaks (DSBs). Impaired function of this kinase leads to a multisystemic disorder including immunodeficiency, progressive cerebellar degeneration, radiation sensitivity, dilated blood vessels, premature aging and a predisposition to cancer.

Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C.

Bernard-Soulier syndrome (BSS) is a rare congenital disease characterized by macrothrombocytopenia and frequent bleeding. It is caused by pathogenic variants in three genes (, or ) that encode for the GPIbα, GPIbβ, and GPIX subunits of the GPIb-V-IX complex, the main platelet surface receptor for von Willebrand factor, being essential for platelet adhesion and aggregation. According to the affected gene, we distinguish BSS type A1 (), type B (), or type C ().

Influenza A virus infection instructs hematopoiesis to megakaryocyte-lineage output.

Respiratory tract infections are among the deadliest communicable diseases worldwide. Severe cases of viral lung infections are often associated with a cytokine storm and alternating platelet numbers. We report that hematopoietic stem and progenitor cells (HSPCs) sense a non-systemic influenza A virus (IAV) infection via inflammatory cytokines.

Understanding the Role of LFA-1 in Leukocyte Adhesion Deficiency Type I (LAD I): Moving towards Inflammation?

LFA-1 (Lymphocyte function-associated antigen-1) is a heterodimeric integrin (CD11a/CD18) present on the surface of all leukocytes; it is essential for leukocyte recruitment to the site of tissue inflammation, but also for other immunological processes such as T cell activation and formation of the immunological synapse. Absent or dysfunctional expression of LFA-1, caused by mutations in the (integrin subunit beta 2) gene, results in a rare immunodeficiency syndrome known as Leukocyte adhesion deficiency type I (LAD I). Patients suffering from severe LAD I present with recurrent infections of the skin and mucosa, as well as inflammatory symptoms complicating the clinical course of the disease before and after allogeneic hematopoietic stem cell transplantation (alloHSCT); alloHSCT is currently the only established curative treatment option.

Establishment of a novel stable human suspension packaging cell line producing ecotropic retroviral MLV(PVC-211) vectors efficiently transducing murine hematopoietic stem and progenitor cells.

Retroviral vectors derived from murine leukemia virus (MLV) are amongst the most frequently utilized vectors in gene therapy approaches such as the genetic modification of hematopoietic cells. Currently, vector particles are mostly produced employing adherent viral packaging cell lines (VPCs) rendering the scale up of production laborious, and thus cost-intensive. Here, we describe the rapid establishment of a human suspension 293-F cell line derived ecotropic MLV VPC.

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.

Forming megakaryocytes from murine-induced pluripotent stem cells by the inducible overexpression of supporting factors.

Background: Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low.

Signaling properties of murine MPL and MPL mutants after stimulation with thrombopoietin and romiplostim.

Thrombopoietin (THPO) and its receptor myeloproliferative leukemia virus oncogene (MPL) regulate hematopoietic stem cell (HSC) quiescence and maintenance, but also megakaryopoiesis. Thrombocytopenias or aplastic anemias can be treated today with THPO peptide mimetics (romiplostim) or small-molecule THPO receptor agonists (e.g.

High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia.

Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting.

Zika virus infection studies with CD34 hematopoietic and megakaryocyte-erythroid progenitors, red blood cells and platelets.

Background: To date, several cases of transfusion-transmitted ZIKV infections have been confirmed. Multiple studies detected prolonged occurrence of ZIKV viral RNA in whole blood as compared to plasma samples indicating potential ZIKV interaction with hematopoietic cells. Also, infection of cells from the granulocyte/macrophage lineage has been demonstrated.

Hematopoietic Stem Cell Transplantation Restores Naïve T-Cell Populations in -Deficient Mice and in Preemptively Treated Patients With Ataxia-Telangiectasia.

Ataxia-telangiectasia (A-T) is a multisystem disorder with progressive cerebellar ataxia, immunodeficiency, chromosomal instability, and increased cancer susceptibility. Cellular immunodeficiency is based on naïve CD4 and CD8 T-cell lymphopenia. Hematopoietic stem cell transplantation (HSCT) offers a potential to cure immunodeficiency and cancer due to restoration of the lymphopoietic system.

Endothelial-platelet interactions in influenza-induced pneumonia: A potential therapeutic target.

Every year, influenza viruses spread around the world, infecting the respiratory systems of countless humans and animals, causing illness and even death. Severe influenza infection is associated with pulmonary epithelial damage and endothelial dysfunction leading to acute lung injury (ALI). There is evidence that an aggressive cytokine storm and cell damage in lung capillaries as well as endothelial/platelet interactions contribute to vascular leakage, pro-thrombotic milieu and infiltration of immune effector cells.

Endothelial protein C receptor supports hematopoietic stem cell engraftment and expansion in Mpl-deficient mice.

Thrombopoietin (Thpo)/myeloproliferative leukemia virus oncogene (Mpl) signaling controls hematopoietic stem cell (HSC) self-renewal and quiescence; however, how these 2 seemingly opposing functions are controlled is not well understood. By transplantation of lentiviral-transduced hematopoietic cells in the Mpl-deficient mouse model, we addressed whether known or predicted Thpo target genes were able to rescue the Mpl-deficient phenotype of the mice. Among the tested genes, we identified endothelial protein C receptor (Epcr) to expand HSCs with the long-term (LT)-HSC surface phenotype in Mpl mice and to enable secondary transplantation of Mpl-deficient bone marrow (BM).

Sustained and regulated gene expression by Tet-inducible "all-in-one" retroviral vectors containing the HNRPA2B1-CBX3 UCOE.

Genetic modification of induced pluripotent stem (iPS) cells may be necessary for the generation of effector cells for cellular therapies. Hereby, it can be important to induce transgene expression at restricted and defined time windows, especially if it interferes with pluripotency or differentiation. To achieve this, inducible expression systems can be used such as the tetracycline-inducible retroviral vector system, however, retroviral expression can be subjected to epigenetic silencing or to position-effect variegation.

generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome.

Chimeric antigen receptor (CAR) T cells brought substantial benefit to patients with B-cell malignancies. Notwithstanding, CAR T-cell manufacturing requires complex procedures impeding the broad supply chain. Here, we provide evidence that human CD19-CAR T cells can be generated directly using the lentiviral vector CD8-LV specifically targeting human CD8 cells.

Non-Clinical Efficacy and Safety Studies on G1XCGD, a Lentiviral Vector for Ex Vivo Gene Therapy of X-Linked Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a debilitating primary immunodeficiency affecting phagocyte function due to the absence of nicotinamide dinucleotide phosphate (NADPH) oxidase activity. The vast majority of CGD patients in the Western world have mutations within the X-linked CYBB gene encoding for gp91 (NOX2), the redox center of the NADPH oxidase complex (XCGD). Current treatments of XCGD are not entirely satisfactory, and prior attempts at autologous gene therapy using gammaretrovirus vectors did not provide long-term curative effects.

Promises and Challenges in Hematopoietic Stem Cell Gene Therapy.

Hematopoietic stem cell-directed gene therapy (HSC-GT) provides an innovative treatment option for hematological disorders. Gene therapy promises to cure the disease "at the root" and is therefore exceptional in its potential, but also formidable in its challenges, as long-term side effects are hard to predict and clinical experience remains limited. Many excellent reviews on the topic by designated experts in the field of HSC-GT have come forth, elucidating the successes and pitfalls in the various clinical studies.

A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis.

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis.

Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy.

Retroviral vectors have shown their curative potential in clinical trials correcting monogenetic disorders. However, therapeutic benefits were compromised due to vector-induced dysregulation of cellular genes and leukemia development in a subset of patients. Bromodomain and extraterminal domain (BET) proteins act as cellular cofactors that tether the murine leukemia virus (MLV) pre-integration complex to host chromatin via interaction with the MLV integrase (IN) and thereby define the typical gammaretroviral integration distribution.

Targeting expression to megakaryocytes and platelets by lineage-specific lentiviral vectors.

Unlabelled: Essentials Platelet phenotypes can be modified by lentiviral transduction of hematopoietic stem cells. Megakaryocyte-specific lentiviral vectors were tested in vitro and in vivo for restricted expression. The glycoprotein 6 vector expressed almost exclusively in megakaryocytes.

CD30 Receptor-Targeted Lentiviral Vectors for Human Induced Pluripotent Stem Cell-Specific Gene Modification.

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4(high) cells of human iPSC cultures and a human embryonic stem cell line.