Publications by authors named "Marina Cavazzana"

102 Publications

Two Monogenetic Disorders, Activated PI3-Kinase-δ Syndrome 2 and Smith-Magenis Syndrome, in One Patient: Case Report and a Literature Review of Neurodevelopmental Impact in Primary Immunodeficiencies Associated With Disturbed PI3K Signaling.

Front Pediatr 2021 24;9:688022. Epub 2021 Jun 24.

Université de Paris, Imagine Institute, Paris, France.

Activated PI3-kinase-δ syndrome 2 (APDS2) is caused by autosomal dominant mutations in the gene encoding the p85α, p55α, and p50α regulatory subunits. Most diagnosed APDS2 patients carry mutations affecting either the splice donor or splice acceptor sites of exon 11 of the gene responsible for an alternative splice product and a shortened protein. The clinical presentation of APDS2 patients is highly variable, ranging from mild to profound combined immunodeficiency features as massive lymphoproliferation, increased susceptibility to bacterial and viral infections, bronchiectasis, autoimmune manifestations, and occurrence of cancer. Non-immunological features such as growth retardation and neurodevelopmental delay have been reported for APDS2 patients. Here, we describe a patient suffering from an APDS2 associated with a Smith-Magenis syndrome (SMS), a complex genetic disorder affecting, among others, neurological manifestations and review the literature describing neurodevelopmental impacts in APDS2 and other PIDs/monogenetic disorders associated with dysregulated PI3K signaling.
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http://dx.doi.org/10.3389/fped.2021.688022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266209PMC
June 2021

Retrieval of vector integration sites from cell-free DNA.

Nat Med 2021 Jun 17. Epub 2021 Jun 17.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget); IRCCS, San Raffaele Scientific Institute, Milan, Italy.

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.
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http://dx.doi.org/10.1038/s41591-021-01389-4DOI Listing
June 2021

A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors.

Cell Mol Immunol 2021 Jul 11;18(7):1662-1676. Epub 2021 Jun 11.

Université de Paris, Imagine Institute, Laboratory of Human Lymphohematopoiesis, INSERM UMR 1163, Paris, France.

Several obstacles to the production, expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy. In the context of HSCT, delayed naïve T-cell recovery contributes to poor outcomes. A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors (HTLPs), allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus. However, it is challenging to produce HTLPs in the high numbers required to meet clinical needs. Here, we found that adding tumor necrosis factor alpha (TNFα) to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival. This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy (including CAR T-cell therapy).
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http://dx.doi.org/10.1038/s41423-021-00706-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245454PMC
July 2021

Single-cell analysis of FOXP3 deficiencies in humans and mice unmasks intrinsic and extrinsic CD4 T cell perturbations.

Nat Immunol 2021 05 8;22(5):607-619. Epub 2021 Apr 8.

Department of Immunology, Harvard Medical School, Boston, MA, USA.

FOXP3 deficiency in mice and in patients with immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome results in fatal autoimmunity by altering regulatory T (T) cells. CD4 T cells in patients with IPEX syndrome and Foxp3-deficient mice were analyzed by single-cell cytometry and RNA-sequencing, revealing heterogeneous T-like cells, some very similar to normal T cells, others more distant. Conventional T cells showed no widespread activation or helper T cell bias, but a monomorphic disease signature affected all CD4 T cells. This signature proved to be cell extrinsic since it was extinguished in mixed bone marrow chimeric mice and heterozygous mothers of patients with IPEX syndrome. Normal T cells exerted dominant suppression, quenching the disease signature and revealing in mutant T-like cells a small cluster of genes regulated cell-intrinsically by FOXP3, including key homeostatic regulators. We propose a two-step pathogenesis model: cell-intrinsic downregulation of core FOXP3-dependent genes destabilizes T cells, de-repressing systemic mediators that imprint the disease signature on all T cells, furthering T cell dysfunction. Accordingly, interleukin-2 treatment improved the T-like compartment and survival.
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http://dx.doi.org/10.1038/s41590-021-00910-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173714PMC
May 2021

Sustained remission after haploidentical bone marrow transplantation in a child with refractory systemic juvenile idiopathic arthritis.

Pediatr Rheumatol Online J 2021 Mar 12;19(1):27. Epub 2021 Mar 12.

Pediatric Hematology-Immunology and Rheumatology Department, Necker-Enfants-Malades University Hospital, Assistance Publique Hôpitaux de Paris, 149 rue de Sèvres, 75015, Paris, France.

Background: Some patients with systemic juvenile idiopathic arthritis (SJIA) and severe, refractory disease achieved remission through intensive immunosuppressive treatment followed by autologous hematopoietic stem cell transplantation (HSCT). However, disease relapsed in most cases. More recently selected SJIA patients received allogenic HSCT from a HLA-identical sibling or a HLA matched unrelated donor. While most transplanted patients achieved sustained SJIA remission off-treatment, the procedure-related morbidity was high.

Case Report: A girl presented SJIA with a severe disease course since the age of 15 months. She was refractory to the combination of methotrexate and steroids to anti-interleukin (IL)-1, then anti-IL-6, tumor necrosis factor alpha inhibitors, and thalidomide. Given the high disease burden and important treatment-related toxicity the indication for a haploidentical HSCT from her mother was validated, as no HLA matched donor was available. The patient received a T replete bone marrow graft at the age of 3.7 years. Conditioning regimen contained Rituximab, Alemtuzumab, Busulfan, and Fludarabine. Cyclophosphamide at D + 3 and + 4 post HSCT was used for graft-versus-host-disease prophylaxis, followed by Cyclosporin A and Mycophenolate Mofetil. Post HSCT complications included severe infections, grade 3 intestinal graft-versus-host-disease, autoimmune thyroiditis, and immune thrombocytopenia. Three years after HSCT, the child is alive and well, notwithstanding persistent hypothyroidy requiring substitution. Immune thrombocytopenia had resolved. Most importantly, SJIA was in complete remission, off immunosuppressive drugs.

Conclusion: Allogenic HSCT may be a therapeutic option, even with a HLA haplo-identical alternative donor, in patients with inflammatory diseases such as SJIA. Despite increased experience with this treatment, the risk of life-threatening complications restrains its indication to selected patients with severe, refractory disease.
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http://dx.doi.org/10.1186/s12969-021-00523-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953742PMC
March 2021

A combination of cyclophosphamide and interleukin-2 allows CD4+ T cells converted to Tregs to control scurfy syndrome.

Blood 2021 Apr;137(17):2326-2336

Institut Imagine, Université de Paris, INSERM UMR1163, Laboratory of Human Lymphohematopoiesis, Paris, France.

Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by mutations in forkhead box P3 (FOXP3), which lead to the loss of function of regulatory T cells (Tregs) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide (Cy) conditioning and interleukin-2 (IL-2) treatment. This model highlighted the possibility of rescuing scurfy disease after the latter's onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and, as a reporter, a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Tregs. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Tregs. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Tregs capable of controlling severe autoimmune disease.
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http://dx.doi.org/10.1182/blood.2020009187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163490PMC
April 2021

Seletalisib for Activated PI3Kδ Syndromes: Open-Label Phase 1b and Extension Studies.

J Immunol 2020 12 28;205(11):2979-2987. Epub 2020 Oct 28.

Imagine Institute, University of Paris, 75015 Paris, France.

Mutations in two genes can result in activated PI3Kδ syndrome (APDS), a rare immunodeficiency disease with limited therapeutic options. Seletalisib, a potent, selective PI3Kδ inhibitor, was evaluated in patients with APDS1 and APDS2. In the phase 1b study (European Clinical Trials Database 2015-002900-10) patients with genetic and clinical confirmation of APDS1 or APDS2 received 15-25 mg/d seletalisib for 12 wk. Patients could enter an extension study (European Clinical Trials Database 2015-005541). Primary endpoints were safety and tolerability, with exploratory efficacy and immunology endpoints. Seven patients (median age 15 years; APDS1 = 3; APDS2 = 4) received seletalisib; five completed the phase 1b study. For the extension study, four patients entered, one withdrew consent (week 24), three completed ≥84 wk of treatment. In the phase 1b study, patients had improved peripheral lymphadenopathy ( = 2), lung function ( = 1), thrombocyte counts ( = 1), and chronic enteropathy ( = 1). Overall, effects were maintained in the extension. In the phase 1b study, percentages of transitional B cells decreased, naive B cells increased, and senescent CD8 T cells decreased (human cells); effects were generally maintained in the extension. Seletalisib-related adverse events occurred in four of seven patients (phase 1b study: hepatic enzyme increased, dizziness, aphthous ulcer, arthralgia, arthritis, increased appetite, increased weight, restlessness, tendon disorder, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (aphthous ulcer). Serious adverse events occurred in three of seven patients (phase 1b study: hospitalization, colitis, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (stomatitis). Patients with APDS receiving seletalisib had improvements in variable clinical and immunological features, and a favorable risk-benefit profile was maintained for ≤96 wk.
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http://dx.doi.org/10.4049/jimmunol.2000326DOI Listing
December 2020

Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype.

Sci Adv 2020 Feb 12;6(7). Epub 2020 Feb 12.

Laboratory of chromatin and gene regulation during development, INSERM UMR1163, Paris, France.

Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) β chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.
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http://dx.doi.org/10.1126/sciadv.aay9392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015694PMC
February 2020

Vascular access for optimal hematopoietic stem cell collection.

J Clin Apher 2021 Feb 27;36(1):12-19. Epub 2020 Aug 27.

Biotherapy Department, Necker Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France.

Background: Autologous and allogeneic hematopoietic stem cell transplantation of cytokine-mobilized peripheral blood stem cells (PBSCs) is increasingly used to treat patients with hematologic disorders. Different types of vascular access have been exploited for the apheresis procedure, including peripheral veins (PV) and central venous catheter (CVC). In some cases, PV access is unavailable. There are few published data on the efficiency and quality of harvesting with different types of vascular access. This study brings out complications and morbidity of this procedure linked to these different access.

Methods: We performed a comparative, retrospective, single-center study of hematopoietic stem cell collection using these two types of vascular access. We compared the efficiency and complication rate for 617 adults apheresis sessions in 401 patients and healthy donors, for PBSC collection via PV or CVC between 2010 and 2016. The quality of the HSC product was evaluated in terms of the total CD34 + count and neutrophil contamination.

Results: The PV and CVC groups did not differ significantly in terms of the quality of the apheresis product, mean ± SD CD34 + cells collected in PV group was 383.1 ± 402.7 × 10e6 and 298.8 ± 372.7 × 10e6 and the level of neutrophil contamination was 21.0 ± 17.8% in the PV group and 20.6 ± 18.4% in the CVC group. The complication rate did not differ between the two groups.

Conclusion: The type of vascular access for apheresis hematopoietic stem cell harvesting must be determined by trained staff. Successful harvesting can be performed via PV then CVC is not needed or not available.
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http://dx.doi.org/10.1002/jca.21828DOI Listing
February 2021

An Evaluation of Three Ways of Communicating Carrier Status Results to the Parents of Children in a Neonatal Sickle Cell Screening Programme.

Front Pediatr 2020 19;8:300. Epub 2020 Jun 19.

Centre d'Investigation Clinique de Biothérapie, Hôpital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Paris, France.

Sickle cell disease (SCD) is the most frequent monogenic disease worldwide; ~5-7% of the world population carry a hemoglobin disorder trait. In the US, one in every 1,941 newborns has SCD, whereas one in every 3,000 newborns in France is affected - resulting in 385 new cases and 5,883 newly identified carriers per year. The objective of the present study was to evaluate three different ways of providing information to parents at risk of having a child with SCD, with a view to increasing the parental screening rate and decreasing the number of new cases per year in France. In a randomized study, we contacted 300 couples of parents after their child had been identified as a SCD carrier in the French national newborn screening programme: 100 couples received an information letter (the standard procedure in France: arm A), 100 couples received a letter and then a follow-up phone call (arm B), and 100 received a letter and then three follow-up text messages at 5-day intervals (arm C). The primary endpoint was the number of parents in each arm screened in the 120 days after the letter had been sent. In a modified intention-to-treat analysis, the screening rate was 17% in arm A, 35% in arm B, and 30% in arm C. Telephone and text message follow-ups were associated with higher screening rates, compared with no follow-up. After being informed of their child's carrier status, some parents had consulted a healthcare professional but had not been referred for screening (16% in arm A, 19% in arm B, and 13% in arm C). A letter followed by a phone call or three text messages is more effective than a letter alone for informing parents at risk of having a child with SCD. The effective implementation of this follow-up programme probably requires better training of all the healthcare professionals involved.
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http://dx.doi.org/10.3389/fped.2020.00300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318296PMC
June 2020

Alternative Promoter Encodes a Functional Munc13-4 Isoform Predominantly Expressed in Lymphocytes and Platelets.

Front Immunol 2020 9;11:1154. Epub 2020 Jun 9.

Department of Medicine, Center for Hematology and Regenerative Medicine, Karolinska Institutet, Stockholm, Sweden.

Autosomal recessive mutations in genes required for cytotoxicity are causative of a life-threatening, early-onset hyperinflammatory syndrome termed familial hemophagocytic lymphohistiocytosis (FHL). Mutations in cause FHL type 3. encodes Munc13-4, a member of the Unc13 protein family which control SNARE complex formation and vesicle fusion. We have previously identified FHL3-associated mutations in the first intron of which control transcription from an alternative transcriptional start site. Using isoform specific antibodies, we demonstrate that this alternative Munc13-4 isoform with a unique N-terminus is preferentially expressed in human lymphocytes and platelets, as compared to the conventional isoform that was mostly expressed in monocytes and neutrophils. The distinct N-terminal of the two isoforms did not impact on Munc13-4 localization or trafficking to the immunological synapse of cytotoxic T cells. Moreover, ectopic expression of both isoforms efficiently restored exocytosis by FHL3 patient-derived Munc13-4 deficient T cells. Thus, we demonstrate that the conventional and alternative Munc13-4 isoforms have different expression pattern in hematopoietic cell subsets, but display similar localization and contribution to T cell exocytosis. The use of an alternative transcriptional starting site (TSS) in lymphocytes and platelets could be selected for increasing the overall levels of Munc13-4 expression for efficient secretory granule release.
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http://dx.doi.org/10.3389/fimmu.2020.01154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296141PMC
April 2021

Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID.

Mol Ther Methods Clin Dev 2020 Jun 31;17:666-682. Epub 2020 Mar 31.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333ZA Leiden, the Netherlands.

Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized to ensure optimal expression. We used mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with expression. Mice with low expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34 cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID.
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http://dx.doi.org/10.1016/j.omtm.2020.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163047PMC
June 2020

Donor-targeted serotherapy as a rescue therapy for steroid-resistant acute GVHD after HLA-mismatched kidney transplantation.

Am J Transplant 2020 08 10;20(8):2243-2253. Epub 2020 Mar 10.

Etablissement Français du Sang, St. Denis, France.

Acute graft-versus-host disease (GVHD) is a rare but frequently lethal complication after solid organ transplantation. GVHD occurs in unduly immunocompromised hosts but requires the escalation of immunosuppression, which does not discriminate between host and donor cells. In contrast, donor-targeted therapy would ideally mitigate graft-versus-host reactivity while sparing recipient immune functions. We report two children with end-stage renal disease and severe primary immune deficiency (Schimke syndrome) who developed severe steroid-resistant acute GVHD along with full and sustained donor T cell chimerism after isolated kidney transplantation. Facing a therapeutic dead end, we used a novel strategy based on the adoptive transfer of anti-HLA donor-specific antibodies (DSAs) through the transfusion of highly selected plasma. After approval by the appropriate regulatory authority, an urgent nationwide search was launched among more than 3800 registered blood donors with known anti-HLA sensitization. Adoptively transferred DSAs bound to and selectively depleted circulating donor T cells. The administration of DSA-rich plasma was well tolerated and notably did not induce antibody-mediated rejection of the renal allografts. Acute GVHD symptoms promptly resolved in one child. This report provides a proof of concept for a highly targeted novel therapeutic approach for solid organ transplantation-associated GVHD.
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http://dx.doi.org/10.1111/ajt.15827DOI Listing
August 2020

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs.

Blood 2020 04;135(15):1219-1231

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA.

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.
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http://dx.doi.org/10.1182/blood.2019002350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146019PMC
April 2020

A gain-of-function RAC2 mutation is associated with bone-marrow hypoplasia and an autosomal dominant form of severe combined immunodeficiency.

Haematologica 2021 02 1;106(2):404-411. Epub 2021 Feb 1.

Biotherapy Clinical Investigation Center, Groupe Hospitalier Universitaire Ouest,Assistance Publique.

Severe combined immunodeficiencies (SCIDs) constitute a heterogeneous group of life-threatening genetic disorders that typically present in the first year of life. They are defined by the absence of autologous T cells and the presence of an intrinsic or extrinsic defect in the B-cell compartment. In three newborns presenting with frequent infections and profound leukopenia, we identified a private, heterozygous mutation in the RAC2 gene (p.G12R). This mutation was de novo in the index case, who had been cured by hematopoietic stem cell transplantation but had transmitted the mutation to her sick daughter. Biochemical assays showed that the mutation was associated with a gain of function. The results of in vitro differentiation assays showed that RAC2 is essential for the survival and differentiation of hematopoietic stem/progenitor cells. Therefore, screening for RAC2 gain-of-function mutations should be considered in patients with a SCID phenotype and who lack a molecular diagnosis.
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http://dx.doi.org/10.3324/haematol.2019.230250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849581PMC
February 2021

Bone Marrow Transplantation in Congenital Erythropoietic Porphyria: Sustained Efficacy but Unexpected Liver Dysfunction.

Biol Blood Marrow Transplant 2020 04 14;26(4):704-711. Epub 2019 Dec 14.

Pediatric Immuno-Hematology and Rheumatology Unit, Necker Enfants Malades Hospital, Assistance Publique-Hôpitaux de Paris, AP-HP, Paris, France.

Congenital erythropoietic porphyria (CEP) is a rare disease characterized by erosive photosensitivity and chronic hemolysis due to a defect of the enzyme uroporphyrinogen-III-synthase (UROS). To date, hematopoietic stem cell transplantation (HSCT) is the only curative therapy for the devastating early and severe form of the disease. We describe 6 patients with CEP treated with HSCT (3 of them twice after failure of a first graft) between 1994 and 2016 in our center, including 2 of the very first living patients treated more than 20 years ago. Four patients are doing well at 6 to 25 years post-HSCT, with near-normal biochemical parameters of porphyrin metabolism without the cutaneous or hematologic features of CEP. One patient died within the first year after HSCT from severe graft-versus-host disease (GVHD), and 1 child died of unexplained acute hepatic failure at 1 year after HSCT, despite full donor chimerism. Retrospectively, it appears that all but 1 child had increased transaminase activity with onset from the early postnatal period, which was significantly more marked in the child who died of liver failure. In contrast, liver function values progressively normalized after engraftment in all other children. Liver pathology before HSCT for 3 patients revealed varying degrees of portal, centrilobular, and perisinusoidal fibrosis; clarification of hepatocytes; and cytosolic porphyrin deposits. The liver porphyrin content in biopsy specimens was >60 times the normal values. Despite difficult engraftment, the long-term efficacy of HSCT in CEP appears to be favorable and reinforces its benefits for the severe form of CEP. Hepatic involvement requires careful evaluation before and after HSCT and further investigation into its pathophysiology and care.
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http://dx.doi.org/10.1016/j.bbmt.2019.12.005DOI Listing
April 2020

Autologous Stem-Cell-Based Gene Therapy for Inherited Disorders: State of the Art and Perspectives.

Front Pediatr 2019 31;7:443. Epub 2019 Oct 31.

Biotherapy Department, Necker Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France.

Gene therapy using patient's own stem cells is rapidly becoming an alternative to allogeneic stem cell transplantation, especially when suitably compatible donors cannot be found. The advent of efficient virus-based methods for delivering therapeutic genes has enabled the development of genetic medicines for inherited disorders of the immune system, hemoglobinopathies, and a number of devastating metabolic diseases. Here, we briefly review the state of the art in the field, including gene editing approaches. A growing number of pediatric diseases can be successfully cured by hematopoietic stem-cell-based gene therapy.
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http://dx.doi.org/10.3389/fped.2019.00443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834641PMC
October 2019

Biosafety Studies of a Clinically Applicable Lentiviral Vector for the Gene Therapy of Artemis-SCID.

Mol Ther Methods Clin Dev 2019 Dec 13;15:232-245. Epub 2019 Sep 13.

Genethon and UMR_S951, INSERM, Université Evry, Université Paris Saclay, Evry, 91002 Evry, France.

Genetic deficiency of the nuclease DCLRE1C/Artemis causes radiosensitive severe combined immunodeficiency (RS-SCID) with lack of peripheral T and B cells and increased sensitivity to ionizing radiations. Gene therapy based on transplanting autologous gene-modified hematopoietic stem cells could significantly improve the health of patients with RS-SCID by correcting their immune system. A lentiviral vector expressing physiological levels of human ARTEMIS mRNA from an EF1a promoter without post-transcriptional regulation was developed as a safe clinically applicable candidate for RS-SCID gene therapy. The vector was purified in GMP-comparable conditions and was not toxic or . Long-term engraftment of vector-transduced hematopoietic cells was achieved in irradiated Artemis-deficient mice following primary and secondary transplantation (6 months each). Vector-treated mice displayed T and B lymphopoiesis and polyclonal T cells, had structured lymphoid tissues, and produced immunoglobulins. Benign signs of inflammation were noted following secondary transplants, likely a feature of the model. There was no evidence of transgene toxicity and no induction of hematopoietic malignancy. , the vector had low genotoxic potential on murine hematopoietic progenitor cells using an immortalization assay. Altogether, these preclinical data show safety and efficacy, and support further development of the vector for the gene therapy of RS-SCID.
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http://dx.doi.org/10.1016/j.omtm.2019.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838972PMC
December 2019

Extensive multilineage analysis in patients with mixed chimerism after allogeneic transplantation for sickle cell disease: insight into hematopoiesis and engraftment thresholds for gene therapy.

Haematologica 2020 05 19;105(5):1240-1247. Epub 2019 Sep 19.

Department of Biotherapy, Necker-Enfants Malades University Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism <50% had hemolysis (reticulocytosis) and higher HbS than their donor. Four of them had donor chimerism <30%, including a patient with AA donor (hemoglobin >10 g/dL) and three with AS donors (hemoglobin <10 g/dL). However, only one vaso-occlusive crisis occurred with 68.7% HbS. Except in the patients with the lowest chimerism, the donor engraftment was lower for T cells than for the other lineages. In a context of mixed chimerism after hematopoietic stem cell transplantation for SCD, myeloid (rather than T cell) engraftment was the key efficacy criterion. Results show that myeloid chimerism as low as 30% was sufficient to prevent a vaso-occlusive crisis in transplants from an AA donor but not constantly from an AS donor. However, the correction of hemolysis requires higher donor chimerism levels ( ≥50%) in both AA and AS recipients. In the future, this group of patients may need a different therapeutic approach.
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http://dx.doi.org/10.3324/haematol.2019.227561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193509PMC
May 2020

Lentiviral and genome-editing strategies for the treatment of β-hemoglobinopathies.

Blood 2019 10;134(15):1203-1213

Biotherapy Department, Necker Children's Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France.

β-Thalassemia and sickle cell disease (SCD) are the most prevalent monogenic diseases. These disorders are caused by quantitative or qualitative defects in the production of adult hemoglobin. Gene therapy is a potential treatment option for patients lacking an allogenic compatible hematopoietic stem cell (HSC) donor. New-generation lentiviral vectors (LVs) carrying a β-globin-like gene have revolutionized this field by allowing effective HSC transduction, with no evidence of genotoxicity to date. Several clinical trials with different types of vector are underway worldwide; the initial results are encouraging with regard to the sustained production of therapeutic hemoglobin, improved biological parameters, a lower transfusion requirement, and better quality of life. Long-term follow-up studies will confirm the safety of LV-based gene therapy. The optimization of patient conditioning, HSC harvesting, and HSC transduction has further improved the therapeutic potential of this approach. Novel LV-based strategies for reactivating endogenous fetal hemoglobin (HbF) are also promising, because elevated HbF levels can reduce the severity of both β-thalassemia and SCD. Lastly, genome-editing approaches designed to correct the disease-causing mutation or reactivate HbF are currently under investigation. Here, we discuss the clinical outcomes of current LV-based gene addition trials and the promising advantages of novel alternative therapeutic strategies.
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http://dx.doi.org/10.1182/blood.2019000949DOI Listing
October 2019

Ex vivo generated human T-lymphoid progenitors as a tool to accelerate immune reconstitution after partially HLA compatible hematopoietic stem cell transplantation or after gene therapy.

Bone Marrow Transplant 2019 08;54(Suppl 2):749-755

Biotherapy Clinical Investigation Center, Groupe Hospitalier Universitaire Ouest, Assistance Publique-Hôpitaux de Paris, INSERM CIC 1416, Paris, France.

Prolonged T-cell immunodeficiency following HLA- incompatible hematopoietic stem cell transplantation (HSCT) represents a major obstacle hampering the more widespread use of this approach. Strategies to fasten T-cell reconstitution in this setting are highly warranted as opportunistic infections and an increased risk of relapse account for high rates of morbidity and mortality especially during early month following this type of HSCT. We have implemented a feeder free cell system based on the use of the notch ligand DL4 and cytokines allowing for the in vitro differentiation of human T-Lymphoid Progenitor cells (HTLPs) from various sources of CD34+ hematopoietic stem and precursor cells (HSPCs). Co- transplantion of human T-lymphoid progenitors (HTLPs) and non- manipulated HSPCs into immunodeficient mice successfully accelerated the reconstitution of a polyclonal T-cell repertoire. This review summarizes preclinical data on the use of T-cell progenitors for treatment of post- transplantation immunodeficiency and gives insights into the development of GMP based protocols for potential clinical applications including gene therapy approaches. Future clinical trials implementing this protocol will aim at the acceleration of immune reconstitution in different clinical settings such as SCID and leukemia patients undergoing allogeneic transplantation. Apart from pure cell-therapy approaches, the combination of DL-4 culture with gene transduction protocols will open new perspectives in terms of gene therapy applications for primary immunodeficiencies.
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http://dx.doi.org/10.1038/s41409-019-0599-9DOI Listing
August 2019

Gene therapy of hemoglobinopathies: progress and future challenges.

Hum Mol Genet 2019 10;28(R1):R24-R30

Paris Descartes-Sorbonne Paris Cité University, Imagine Institute, Paris, France.

Recently, gene therapy clinical trials have been successfully applied to hemoglobinopathies, such as sickle cell disease (SCD) and β-thalassemia. Among the great discoveries that led to the design of genetic approaches to cure these disorders is the discovery of the β-globin locus control region and several associated transcription factors, which determine hemoglobin switching as well as high-level, erythroid-specific expression of genes at the ß-globin locus. Moreover, increasing evidence shows that lentiviral vectors are efficient tools to insert large DNA elements into nondividing hematopoietic stem cells, showing reassuring safe integration profiles. Alternatively, genome editing could restore expression of fetal hemoglobin or target specific mutations to restore expression of the wild-type β-globin gene. The most recent clinical trials for β-thalassemia and SCD are showing promising outcomes: patients were able to discontinue transfusions or had reduced transfusion requirements. However, toxic myeloablation and the high cost of current ex vivo hematopoietic stem cell gene therapy platforms represent a barrier to a widespread application of these approaches. In this review, we summarize these gene therapy strategies and ongoing clinical trials. Finally, we discuss possible strategies to improve outcomes, reduce myeloablative regimens and future challenges to reduce the cost of gene therapy platform.
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http://dx.doi.org/10.1093/hmg/ddz172DOI Listing
October 2019

Safety of CD34 Hematopoietic Stem Cells and CD4 T Lymphocytes Transduced with LVsh5/C46 in HIV-1 Infected Patients with High-Risk Lymphoma.

Mol Ther Methods Clin Dev 2019 Jun 26;13:303-309. Epub 2019 Feb 26.

Paris Descartes, Sorbonne Paris Cité, France.

Although the risk of developing lymphoma has decreased in the highly active antiretroviral therapy era, this cancer remains the major cause of mortality in HIV-infected patients. Autologous hematopoietic stem cell transplantation (ASCT) outcome does not differ for HIV-infected versus HIV-uninfected patients. We propose to develop a new treatment for HIV-associated high-risk lymphoma based on autologous transplantation of two genetically modified products: CD4 T lymphocytes and CD34 hematopoietic stem cells (HSPCs). The cells will be transduced with the Cal-1 lentiviral vector encoding for both a short hairpin RNA (shRNA) against CCR5 (sh5) and the HIV-1 fusion inhibitor C46. The transduced cells will be resistant to HIV infection by two complementary mechanisms: impaired binding of the virus to the cellular CCR5 co-receptor and decreased fusion of the virus as C46 interacts with gp41 and inhibits HIV infection. This phase I/II pilot study, also entitled GENHIV, will involve two French participating centers: Saint Louis Hospital and Necker Hospital in Paris. We plan to enroll five HIV-1-infected patients presenting with high-risk lymphoma and require a treatment with ASCT. The primary objective of this study is to evaluate the safety, feasibility, and success of engraftment of Cal-1 gene-transduced CD4 T lymphocytes and CD34 HSPCs.
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http://dx.doi.org/10.1016/j.omtm.2019.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416524PMC
June 2019

Concise Review: Boosting T-Cell Reconstitution Following Allogeneic Transplantation-Current Concepts and Future Perspectives.

Stem Cells Transl Med 2019 07 18;8(7):650-657. Epub 2019 Mar 18.

Laboratory of Human Lymphohematopoiesis, INSERM UMR 1163, Imagine Institute, Paris, France.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for a large number of malignant and nonmalignant (inherited) diseases of the hematopoietic system. Nevertheless, non-HLA identical transplantations are complicated by a severe T-cell immunodeficiency associated with a high rate of infection, relapse and graft-versus-host disease. Initial recovery of T-cell immunity following HSCT relies on peripheral expansion of memory T cells mostly driven by cytokines. The reconstitution of a diverse, self-tolerant, and naive T-cell repertoire, however, may take up to 2 years and crucially relies on the interaction of T-cell progenitors with the host thymic epithelium, which may be altered by GvHD, age or transplant-related toxicities. In this review, we summarize current concepts to stimulate reconstitution of a peripheral and polyclonal T-cell compartment following allogeneic transplantation such as graft manipulation (i.e., T-cell depletion), transfusion of ex vivo manipulated donor T cells or the exogenous administration of cytokines and growth factors to stimulate host-thymopoiesis with emphasis on approaches which have led to clinical trials. Particular attention will be given to the development of cellular therapies such as the ex vivo generation of T-cell precursors to fasten generation of a polyclonal and functional host-derived T-cell repertoire. Having been tested so far only in preclinical mouse models, clinical studies are now on the way to validate the efficacy of such T-cell progenitors in enhancing immune reconstitution following HSCT in various clinical settings. Stem Cells Translational Medicine 2019;00:1-8.
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http://dx.doi.org/10.1002/sctm.18-0248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591542PMC
July 2019

Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges.

Nat Rev Drug Discov 2019 06;18(6):447-462

INSERM UMR 1163, Laboratory of Human Lymphohematopoiesis, Paris, France.

Pioneering gene therapy trials have shown that the genetic engineering of haematopoietic stem and progenitor cells can be an alternative to allogeneic transplantation in the treatment of primary immunodeficiencies. Early trials also highlighted the risk of insertional mutagenesis and oncogene transactivation associated with the first generation of gammaretroviral vectors. These events prompted the development of safer, self-inactivating lentiviral or gammaretroviral vectors. These lentiviral vectors have been successfully used to treat over 200 patients with 10 different haematological disorders (including primary immunodeficiencies, haemoglobinopathies and metabolic disorders) and for the generation of chimeric antigen receptor-T cells for cancer therapy. However, several challenges, such as effective reconstitution during inflammation, remain if gene therapy is to be extended to more complex diseases in which haematopoietic stem and progenitor cells can be altered by the disease environment. We discuss the progress made and future challenges for gene therapy and contrast gene therapy with gene-editing strategies.
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http://dx.doi.org/10.1038/s41573-019-0020-9DOI Listing
June 2019

Baboon envelope LVs efficiently transduced human adult, fetal, and progenitor T cells and corrected SCID-X1 T-cell deficiency.

Blood Adv 2019 02;3(3):461-475

Centre International de Recherche en Infectiologie, Université Lyon, Université Claude Bernard Lyon 1, INSERM, U1111, Centre National de la Recherche Scientifique, Unité Mixte de Recherche (UMR) 5308, Ecole Normale Supérieure de Lyon, Lyon, France.

T cells represent a valuable tool for treating cancers and infectious and inherited diseases; however, they are mainly short-lived in vivo. T-cell therapies would strongly benefit from gene transfer into long-lived persisting naive T cells or T-cell progenitors. Here we demonstrate that baboon envelope glycoprotein pseudotyped lentiviral vectors (BaEV-LVs) far outperformed other LV pseudotypes for transduction of naive adult and fetal interleukin-7-stimulated T cells. Remarkably, BaEV-LVs efficiently transduced thymocytes and T-cell progenitors generated by culture of CD34 cells on Delta-like ligand 4 (Dll4). Upon NOD/SCIDγC engraftment, high transduction levels (80%-90%) were maintained in all T-cell subpopulations. Moreover, T-cell lineage reconstitution was accelerated in NOD/SCIDγC recipients after T-cell progenitor injection compared with hematopoietic stem cell transplantation. Furthermore, γC-encoding BaEV-LVs very efficiently transduced Dll4-generated T-cell precursors from a patient with X-linked severe combined immunodeficiency (SCID-X1), which fully rescued T-cell development in vitro. These results indicate that BaEV-LVs are valuable tools for the genetic modification of naive T cells, which are important targets for gene therapy. Moreover, they allowed for the generation of gene-corrected T-cell progenitors that rescued SCID-X1 T-cell development in vitro. Ultimately, the coinjection of LV-corrected T-cell progenitors and hematopoietic stem cells might accelerate T-cell reconstitution in immunodeficient patients.
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http://dx.doi.org/10.1182/bloodadvances.2018027508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373736PMC
February 2019

Innovative Curative Treatment of Beta Thalassemia: Cost-Efficacy Analysis of Gene Therapy Versus Allogenic Hematopoietic Stem-Cell Transplantation.

Hum Gene Ther 2019 06 3;30(6):753-761. Epub 2019 May 3.

1 URC Eco, Assistance Publique-Hôpitaux de Paris, Paris, France.

Seventy-five percent of patients with beta thalassemia (β-thalassemia) do not have human leukocyte antigen-matched siblings and until recently had no access to a curative treatment. Gene therapy is a promising treatment that can be proposed to these patients. This study estimates its cost and efficacy. In a monocentric retrospective study and cost-efficacy analysis, this study compared the two-year outcomes and costs of patients with β-thalassemia treated by gene therapy and hematopoietic stem-cell transplantation (HSCT). Grade III and grade IV complications, hospitalizations, and length of stay were extracted from the hospital discharge data. Costs were estimated from hospital accounting information and national cost studies. A total of seven patients with β-thalassemia treated between 2009 and 2016 were included, of whom four received gene therapy. Patients treated by gene therapy were older and had fewer complications and hospital admissions. Infectious complications were three times more frequent for patients treated with HSCT than for gene therapy. Average costs were €608,086 for patients treated by gene therapy and €215,571 for HSCT. The total cost of the vector was 48% of the total cost of gene therapy. Gene therapy as a curative alternative for patients lacking human leukocyte antigen-matched donors was costlier but resulted in fewer complications than HSCT.
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http://dx.doi.org/10.1089/hum.2018.178DOI Listing
June 2019

Hematopoietic stem cell transplantation for CD40 ligand deficiency: Results from an EBMT/ESID-IEWP-SCETIDE-PIDTC study.

J Allergy Clin Immunol 2019 06 17;143(6):2238-2253. Epub 2019 Jan 17.

Department of Pediatrics, University Medical Center Ulm, Ulm, Germany.

Background: CD40 ligand (CD40L) deficiency, an X-linked primary immunodeficiency, causes recurrent sinopulmonary, Pneumocystis and Cryptosporidium species infections. Long-term survival with supportive therapy is poor. Currently, the only curative treatment is hematopoietic stem cell transplantation (HSCT).

Objective: We performed an international collaborative study to improve patients' management, aiming to individualize risk factors and determine optimal HSCT characteristics.

Methods: We retrospectively collected data on 130 patients who underwent HSCT for CD40L deficiency between 1993-2015. We analyzed outcome and variables' relevance with respect to survival and cure.

Results: Overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were 78.2%, 58.1%, and 72.3% 5 years after HSCT. Results were better in transplantations performed in 2000 or later and in children less than 10 years old at the time of HSCT. Pre-existing organ damage negatively influenced outcome. Sclerosing cholangitis was the most important risk factor. After 2000, superior OS was achieved with matched donors. Use of myeloablative regimens and HSCT at 2 years or less from diagnosis associated with higher OS and DFS. EFS was best with matched sibling donors, myeloablative conditioning (MAC), and bone marrow-derived stem cells. Most rejections occurred after reduced-intensity or nonmyeloablative conditioning, which associated with poor donor cell engraftment. Mortality occurred mainly early after HSCT, predominantly from infections. Among survivors who ceased immunoglobulin replacement, T-lymphocyte chimerism was 50% or greater donor in 85.2%.

Conclusion: HSCT is curative in patients with CD40L deficiency, with improved outcome if performed before organ damage development. MAC is associated with better OS, EFS, and DFS. Prospective studies are required to compare the risks of HSCT with those of lifelong supportive therapy.
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http://dx.doi.org/10.1016/j.jaci.2018.12.1010DOI Listing
June 2019