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. Since allogenic hematopoietic stem cell (HSC) transplantation improved disease outcome, gene therapy based on autologous HSCs is an alternative promising concept. However, due to the large cDNA of ATM (9.2 kb), efficient packaging of retroviral particles and sufficient transduction of HSCs remains challenging.We generated lentiviral, gammaretroviral and foamy viral vectors with a GFP.F2A.Atm fusion or a GFP transgene and systematically compared transduction efficiencies. Vector titers dropped with increasing transgene size, but despite their described limited packaging capacity, we were able to produce lentiviral and gammaretroviral particles. The reduction in titers could not be explained by impaired packaging of the viral genomes, but the main differences occurred after transduction. Finally, after transduction of Atm-deficient (ATM-KO) murine fibroblasts with the lentiviral vector expressing Atm, we could show the expression of ATM protein which phosphorylated its downstream substrates (pKap1 and p-p53).

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 (GP1BA, GP1BB, or GP9) that encode for the GPIbα, GPIbß, 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 (GP1BA), type B (GP1BB), or type C (GP9). Pathogenic variants in these genes cause absent, incomplete, or dysfunctional GPIb-V-IX receptor and, consequently, a hemorrhagic phenotype. Using gene-editing tools, we generated knockout (KO) human cellular models that helped us to better understand GPIb-V-IX complex assembly. Furthermore, we developed novel lentiviral vectors capable of correcting GPIX expression, localization, and functionality in human GP9-KO megakaryoblastic cell lines. Generated GP9-KO induced pluripotent stem cells produced platelets that recapitulated the BSS phenotype: absence of GPIX on the membrane surface and large size. Importantly, gene therapy tools reverted both characteristics. Finally, hematopoietic stem cells from two unrelated BSS type C patients were transduced with the gene therapy vectors and differentiated to produce GPIX-expressing megakaryocytes and platelets with a reduced size. These results demonstrate the potential of lentiviral-based gene therapy to rescue BSS 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. Irrespective of antiviral treatment or vaccination, at a certain threshold of IAV titer in the lung, CD41-positive hematopoietic stem cells (HSCs) enter the cell cycle while endothelial protein C receptor-positive CD41-negative HSCs remain quiescent. Active CD41-positive HSCs represent the source of megakaryocytes, while their multi-lineage reconstitution potential is reduced. This emergency megakaryopoiesis is thrombopoietin independent and attenuated in IAV-infected interleukin-1 receptor-deficient mice. Newly produced platelets during IAV infection are immature and hyper-reactive. After viral clearance, HSC quiescence is re-established. Our study reveals that non-systemic viral respiratory infection has an acute impact on HSCs via inflammatory cytokines to counteract IAV-induced thrombocytopenia.

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 ITGB2 (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. With this review, we aim to provide an overview of the intrinsic role of inflammation in LAD I.

Targeting transgenic proteins to alpha granules for platelet-directed gene therapy.

Platelets are anucleate blood cells that are shed from megakaryocytes (MKs) into the bloodstream to maintain hemostasis and promote wound healing after vascular injury. To carry out their functions, platelets become activated and release bioactive substances from their secretory granules. As alpha granules (αGs) in resting platelets store proteins and release them only after activation, the packaging of proteins into αGs is an attractive strategy to deliver therapeutic proteins. Here, we propose an adjustable model for targeting transgenic proteins to platelet αGs using third-generation self-inactivating lentiviral vectors. The vectors express from the murine platelet factor 4 promoter (mPf4P), restricting transgene expression to the MK lineage. For the delivery and retention of expressed proteins in αGs, proteins are fused to short peptide sorting signals derived from the human cytokine RANTES or from the transmembrane protein P-selectin. We demonstrate effective targeting of GFP to αGs of murine and human in vitro-differentiated MKs and murine platelets in vivo. Furthermore, interferon-α (IFNα), as a potentially therapeutic cytokine, was successfully delivered to and stored in murine platelets in vivo, was released after activation, and inhibited virus replication in vitro. Our vectors create possibilities for numerous applications in cell therapy utilizing platelets as carriers of therapeutic proteins.

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. Using transposon vector technology, a packaging and envelope expression cassette as well as a transfer vector facilitated the establishment of a stable VPC yielding high titers of up to 5.2 × 106 transducing units/mL (TU/mL). Vectors were concentrated using ultrafiltration devices and upon one freeze-thaw-cycle still routinely yielded titers of > 1 × 106 TU/mL. Formation of replication-competent retroviruses was not detected. However and as a first generation transfer vector was used in this proof-of-concept (POC) study, gag gene sequences were transduced into target cells within a range of 1-10 copies per 1000 genomes indicating the homologous recombination of packaging construct elements with the transfer vector. High yield VPC vector productivity was stable over a couple of months and unintended integration of the transposase gene was not observed. Ecotropic MLV vector particles were demonstrated to efficiently transduce primary murine hematopoietic stem and progenitor cells. This novel concept should foster the future establishment of suspension VPCs.

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.

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. OBJECTIVES: To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA-binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre-B-cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). METHODS: To avoid off-target effects, we generated iPSCs carrying the reverse tetracycline-responsive transactivator M2 (rtTA-M2) in the Rosa26 locus and expressed the factors from Tet-inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. RESULTS: Overexpression of GATA1 and Pbx1 increased MK output 2- to 2.5-fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro-generated platelets were functional in spreading on fibrinogen or collagen-related peptide. CONCLUSION: We demonstrate that the use of rtTA-M2 transgenic iPSCs transduced with Tet-inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.

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., eltrombopag). These THPO mimetics were designed for human application; however, many preclinical studies are performed in murine models. We investigated the activation of wild-type murine MPL (mMPL) by romiplostim. Romiplostim stimulated AKT, ERK1/2, and STAT5 phosphorylation without preference for one of these pathways, however, with a four- to fivefold lower phosphorylation intensity at high concentration. Faster internalization of mMPL after romiplostim binding could be one explanation of reduced signaling. In vitro megakaryocyte differentiation, proliferation, and maturation by romiplostim was less efficient compared with stimulation with mTHPO. We further dissected mMPL signaling by lentiviral overexpression of mMPL mutants with tyrosine (Y)-to-phenylalanine (F) substitutions in the distal cytoplasmic tyrosines 582 (Y582F), 616 (Y616F), and 621 (Y621F) individually and in combination (Y616F_Y621F) and in truncated receptors lacking 53 (Δ53) or 69 (Δ69) C-terminal amino acids. Mutation at tyrosine residue Y582F caused a gain-of-function with baseline activation and increased ERK1/2 phosphorylation upon stimulation. In agreement with this, proliferation in Y582F-32D cells was increased, yet did not rescue in vitro megakaryopoiesis from Mpl-deficient cells. Y616F and Y621F mutated receptors exhibited strongly impaired ERK1/2 and decreased AKT signaling and conferred reduced proliferation to 32D cells upon mTHPO stimulation but a partial correction of immature megakaryopoiesis in vitro.

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. Patients may develop severe thrombocytopenia, microcytic anemia, and a fatal course of disease occurred in a patient with sickle cell anemia suggesting additional interference of ZIKV with erythroid and megakaryocytic cells. Therefore, we analyzed whether ZIKV propagates in or compartmentalizes with hematopoietic progenitor, erythroid, and megakaryocytic cells. METHODS: ZIKV RNA replication, protein translation and infectious particle formation in hematopoietic cell lines as well as primary CD34+ HSPCs and ex vivo differentiated erythroid and megakaryocytic cells was monitored using qRT-PCR, FACS, immunofluorescence analysis and infectivity assays. Distribution of ZIKV RNA and infectious particles in spiked red blood cell (RBC) units or platelet concentrates (PCs) was evaluated. RESULTS: While subsets of K562 and KU812Ep6EPO cells supported ZIKV propagation, primary CD34+ HSPCs, MEP cells, RBCs, and platelets were non-permissive for ZIKV infection. In spiking studies, ZIKV RNA was detectable for 7 days in all fractions of RBC units and PCs, however, ZIKV infectious particles were not associated with erythrocytes or platelets. CONCLUSION: Viral particles from plasma or contaminating leukocytes, rather than purified CD34+ HSPCs or the cellular component of RBC units or PCs, present the greatest risk for transfusion-transmitted ZIKV infections.

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. To date, treatments for ALI caused by influenza are limited to antiviral drugs, active ventilation or further symptomatic treatments. In this review, we summarize the mechanisms of influenza-mediated pathogenesis, permissive animal models and histopathological changes of lung tissue in both mice and men and compare it with histological and electron microscopic data from our own group. We highlight the molecular and cellular interactions between pulmonary endothelium and platelets in homeostasis and influenza-induced pathogenesis. Finally, we discuss novel therapeutic targets on platelets/endothelial interaction to reduce or resolve ALI.

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

Background: 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. The aim of this investigation was to analyze the effect of HSCT on naïve CD4+ as well as CD8+ T-cell numbers in A-T. Methods: We analyzed total numbers of peripheral naïve (CD45RA+CD62L+) and memory (CD45RO+CD62L-) CD4+ and CD8+ T-cells of 32 A-T patients. Naïve (CD62LhighCD44low) and memory (CD62LlowCD44high) T-cells were also measured in Atm-deficient mice before and after HSCT with GFP-expressing bone marrow derived hematopoietic stem cells. In addition, we analyzed T-cells in the peripheral blood of two A-T patients after HLA-identic allogeneic HSCT. Results: Like in humans, naïve CD4+ as well as naïve CD8+ lymphocytes were decreased in Atm-deficient mice. HSCT significantly inhibited thymic lymphomas and increased survival time in these animals. Donor cell chimerism increased up to more than 50% 6 months after HSCT accompanied by a significant increase of naïve CD4 and CD8 T-cell subpopulations, but not of memory T-cells. This finding was also identified in the blood of the A-T patients after HSCT. Conclusion: HSCT seems to be a feasible strategy to overcome immunodeficiency and might be a conceivable strategy to avoid T-cell driven cancer in A-T at higher risk for malignancy. Naïve CD4 and CD8 T-cells counts are suitable markers for monitoring immune reconstitution post-HSCT. However, risks and benefits of HSCT in A-T have to be properly weighted.

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). Epcr-transduced Mpl-/- HSCs enter quiescence earlier after transplantation than control-transduced Mpl-/- cells, and upregulated expression of the anti-apoptotic gene Bcl-xL. Also, in the wild-type background, Epcr expression marked the engrafting population in the BM. Furthermore, Epcr expression in Mpl-/- hematopoiesis increased the number of megakaryocytes in the BM. In vitro Thpo supported the surface expression of Epcr on primary murine hematopoietic stem and progenitor cells. With these data, we add new insights into Thpo-dependent influence on HSC engraftment after transplantation. This may be of use for the in vitro manipulation of HSCs, also in the context of gene therapy.

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. One strategy to overcome this is the incorporation of ubiquitous chromatin opening elements (UCOE®'s) into retroviral vectors to maintain a transcriptionally permissive chromatin state at the integration site. In this study, we developed Tet-inducible all-in-one gammaretroviral vectors carrying different sized UCOE®'s derived from the A2UCOE. The ability to prevent vector silencing by preserving the Tet-regulatory potential was investigated in different cell lines, and in murine and human iPS cells. A 670-bp fragment spanning the CBX3 promoter region of A2UCOE (U670) was the most potent element in preventing silencing, and conferred the strongest expression from the vector in the induced state. While longer fragments of A2UCOEs also sustained expression, vector titers and induction efficiencies were impaired. Finally, we demonstrate that U670 can be used for constitutive expression of the transactivator in the all-in-one vector for faithful regulation of transgenes by doxycycline, including the thrombopoietin receptor Mpl conferring cytokine-dependent cell growth.

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. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield.

In vivo 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 in vivo using the lentiviral vector CD8-LV specifically targeting human CD8+ cells. Administration into mice xenografted with Raji lymphoma cells and human peripheral blood mononuclear cells led to CAR expression solely in CD8+ T cells and efficacious elimination of CD19+ B cells. Further, upon injection of CD8-LV into mice transplanted with human CD34+ cells, induction of CAR T cells and CD19+ B-cell depletion was observed in 7 out of 10 treated animals. Notably, three mice showed elevated levels of human cytokines in plasma. Tissue-invading CAR T cells and complete elimination of the B-lymphocyte-rich zones in spleen were indicative of a cytokine release syndrome. Our data demonstrate the feasibility of in vivo reprogramming of human CD8+ CAR T cells active against CD19+ cells, yet with similar adverse effects currently notorious in the clinical practice.

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 gp91phox (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. A new strategy was developed based on the use of the lentiviral vector G1XCGD expressing high levels of the gp91phox transgene in myeloid cells. As a requisite for a clinical trial approval, standardized non-clinical studies were conducted in vitro and in mice in order to evaluate the pharmacodynamics and biosafety of the vector and the biodistribution of G1XCGD-transduced cells. Transduced CD34+ cells derived from XCGD patients engrafted and differentiated similarly to their non-transduced counterparts in xenograft mouse models and generated therapeutically relevant levels of NADPH activity in myeloid cells expressing gp91phox. Expression of functional gp91phox in hematopoietic cells did not affect their homing properties, which engrafted at high levels in mice. Extensive in vitro and in vivo genotoxicity studies found no evidence for adverse mutagenesis related to vector treatment. These studies paved the way for the approval of clinical trials in Europe and in the United States for the treatment of XCGD patients with G1XCGD gene-modified autologous hematopoietic cells.

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. This review attempts to discuss what we understand from those studies to represent current state of the art with respect to vectors, stem cell transduction, and pretransplant preparatory regimes, what limitations may remain, and which types of diseases may be more suited for HSC-GT than others (targets). We thus discuss the available vector platforms (tools) and preclinical/clinical and basic research (tricks) that contribute to our current understanding of HSC-GT, as well as some overarching principles we can conclude from these. The field has also learned from previous shortcomings, although some of the major concerns of the past, specifically insertional mutagenesis, may not be of relevance for future trials. This very positive development in HSC-GT, however, has to compete with the improvements in hematopoietic stem cell transplantation or enzyme-replacement therapy, leaving a narrow margin for gene therapy.

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. We engineered next-generation BET-independent (Bin) MLV vectors to retarget their integration to regions where they are less likely to dysregulate nearby genes. We mutated MLV IN to uncouple BET protein interaction and fused it with chromatin-binding peptides. The addition of the CBX1 chromodomain to MLV INW390A efficiently targeted integration away from gene regulatory elements. The retargeted vector produced at high titers and efficiently transduced CD34+ hematopoietic stem cells, while fewer colonies were detected in a serial colony-forming assay, a surrogate test for genotoxicity. Our findings underscore the potential of the engineered vectors to reduce the risk of insertional mutagenesis without compromising transduction efficiency. Ultimately, combined with other safety features in vector design, next-generation BinMLV vectors can improve the safety of gammaretroviral vectors for gene therapy.

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. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard-Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.