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Mesenchymal stromal cells (MSCs) have been employed in vitroto support hematopoietic stem and progenitor cell (HSPC) expansion and in vivoto promote HSPC engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize the transplantation outcome of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable of alleviating the proliferation arrest and mitigating the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential in vitro. The use of BM-MSCs resulted in superior human engraftment and increased clonal output of GE-HSPCs contributing to the early phase of hematological reconstitution in the peripheral blood of transplanted mice. In conclusion, our work poses the biological bases for a novel clinical use of BM-MSCs to promote engraftment of GE-HSPCs and improve their transplantation outcome.

Adenosine deaminase 2 deficiency (DADA2) is a rare inherited disorder that is caused by autosomal recessive mutations in the ADA2 gene. Clinical manifestations include early-onset lacunar strokes, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic defects. Anti–tumor necrosis factor therapy reduces strokes and systemic inflammation. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Autologous HSPC gene therapy may be an alternative curative option for patients with DADA2. We designed a lentiviral vector encoding ADA2 (LV-ADA2) to genetically correct HSPCs. Lentiviral transduction allowed efficient delivery of the functional ADA2 enzyme into HSPCs from healthy donors. Supranormal ADA2 expression in human and mouse HSPCs did not affect their multipotency and engraftment potential in vivo. The LV-ADA2 induced stable ADA2 expression and corrected the enzymatic defect in HSPCs derived from DADA2 patients. Patients’ HSPCs re-expressing ADA2 retained their potential to differentiate into erythroid and myeloid cells. Delivery of ADA2 enzymatic activity in patients’ macrophages led to a complete rescue of the exaggerated inflammatory cytokine production. Our data indicate that HSPCs ectopically expressing ADA2 retain their multipotent differentiation ability, leading to functional correction of macrophage defects. Altogether, these findings support the implementation of HSPC gene therapy for DADA2.

Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91phoxexpression in X-linked chronic granulomatous disease (XCGD) mice. We found persistence of gene-corrected cells for up to 1 year, restoration of Gp91phoxexpression and NADPH oxidase activity in XCGD phagocytes, and reduced tissue inflammation after LV-mediated HSPC GT. Although most of the mice showed no hematological or biochemical toxicity, a small subset of XCGD GT mice developed T cell lymphoblastic lymphoma (2.94%) and myeloid leukemia (5.88%). No hematological malignancies were identified in C57BL/6 mice transplanted with transduced XCGD HSPCs. Integration pattern analysis revealed an oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in mice with tumors. Collectively, our data support the long-term efficacy of LV-mediated HSPC GT in XCGD mice and provide a safety warning because the chronic inflammatory XCGD background may contribute to oncogenesis.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1in HSPCs from patients with hypomorphic RAG1mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1–/–mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

•Circulating multilymphoid progenitors are primed for the seeding the thymus and contribute to steady-state T-cell lymphopoiesis.•cHSPCs are responsible for clonal redistribution among BM niches and actively participate in human hematopoiesis.

Background and Rationale

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper–immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α−directed migration. Gene transfer of ARPC1B in patients’ T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1Bhave been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1Bin 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper–immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α−directed migration. Gene transfer of ARPC1Bin patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

Dysregulation of the interleukin-1 (IL-1) pathway leads to immune diseases that can result in chronic tissue and organ inflammation. Although IL-1 blockade has shown promise in ameliorating these symptoms and improving patients’ quality of life, there is an urgent need for more effective, long-lasting treatments. We developed a lentivirus (LV)–mediated gene transfer strategy using transplanted autologous hematopoietic stem/progenitor cells (HSPCs) as a source of IL-1 receptor antagonist (IL-1RA) for systemic delivery to tissues and organs. Transplantation of mouse and human HSPCs transduced with an IL-1RA–encoding LV ensured stable IL-1RA production while maintaining the clonogenic and differentiation capacities of HSPCs in vivo. We examined the efficacy of cell-mediated IL-1RA delivery in three models of IL-1–dependent inflammation, for which treatment hindered neutrophil recruitment in an inducible model of gout, prevented systemic and multi-tissue inflammation in a genetic model of cryopyrin-associated periodic syndromes, and reduced disease severity in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Our findings demonstrate HSPC-mediated IL-1RA delivery as a potential therapeutic modality that can be exploited to suppress tissue and organ inflammation in diverse immune-related diseases involving IL-1–driven inflammation.

Hematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases.

Adenosine deaminase 2 deficiency (DADA2) is a recently defined inborn error of immunity caused by loss-of-function mutations in the ADA2 gene. Patients suffer from severe manifestations, including early-onset lacunar strokes, intracranial hemorrhages, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic abnormalities. The therapeutic benefit of the current treatments is unsatisfactory. Anti-tumor necrosis factor therapy reduces strokes and systemic inflammation but does not correct cytopenia and bone marrow failure. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Therefore, we proposed that autologous HSPC gene therapy may be an alternative curative option for patients who has no compatible donor or cannot receive intense chemotherapy.

From a series of 850 patients with head and neck carcinoma and subjected to lymph node dissection, 80 cases of recurrences in the neck have been collected. Postoperative radiotherapy was performed only in cases with metastatic extranodal spread. Of these recurrences, 56 occurred in the area of lymph node dissection, 7 were marginal and 17 were contralateral. The recurrences occurred prevalently in node-positive (N +) patients (70 of 80). The incidence of recurrences in the dissection area was 41.6 % (25 of 60) in cases with metastatic extranodal spread, despite postoperative radiotherapy. The incidence of recurrences in cases with clinically evident metastases at the time of dissection but without extranodal spread and not subjected to postoperative radiotherapy was relatively high (24.1 %, or 28 of 116). Since recurrences occurred, despite postoperative radiotherapy, in a relatively high percentage of cases with carcinoma of the oral floor and of the tongue (59.1 % and 50 %, respectively), it seems justifiable to perform preoperative radiation treatment in cases with clinically evident metastatic lymph nodes. As regards marginal recurrences, which all occurred in patients with carcinoma of the oral floor, it is considered sufficient to extend the surgical treatment to the subhyoid region. The high incidence of contralateral recurrences, which occurred mainly in patients with carcinoma of the larynx (13 of 17), shows the usefulness of radiation treatment of the contralateral region of the neck in these tumors, when dissection is limited to only one side of the neck.

Forty-two cases of tumors of the skin and of the soft tissues immediately beneath the skin, presumably induced by ionizing radiotherapy, are reported: 35 were carcinomas, 2 angiosarcomas, 2 leiomyosarcomas of the dermis and 3 fibrosarcomas. In 31 of the 35 cases of carcinomas, multiple neoplastic foci were found in the skin area exposed to the ionizing radiation. The median age of the patients at the time of exposure to ionizing radiation was 32.5 years for those with carcinomas and 30 years for the others. The relatively young age at exposure to the ionizing radiation may have influenced onset of the tumor. The median interval between exposure to ionizing radiation and clinical finding of the tumor was 18.1 years for the carcinomas and 16 for the other tumor types. The radiation dose to which the patients had been subjected varied for the carcinomas from 12 to 25 Gy and for the other tumors from 40 to 50 Gy, administered over 1 to 4 years in the cases of carcinomas and 4 to 5 weeks in the other cases. The dose administered to the cases with cutaneous carcinomas was rather low, since almost all these patients had benign disease; however, it is in this dose range (12-25 Gy) that, according to Gray, a relatively high incidence of induced tumors is verified. In the opinion of the author of the present paper, the multiplicity of neoplastic carcinomatous foci in the skin area exposed to radiation confirms Gray's hypothesis; also, the time over which the dose was administered was presumably important in determining such multiplicity. The soft tissue sarcomas occurred only in patients previously subjected to radiotherapy, according to traditional modalities, for malignant neoplasms. The carcinomas were observed almost always in the trunk, and like spontaneous carcinomas at this site they were almost exclusively of basal cell type.

A series of 45 recurrences in the soft tissues of the neck following lymph node dissection in 497 patients bearing carcinoma of the upper aerodigestive passages is reported. Only 22 cases that presented perilymph node metastases and/or in which there were reasons to indicate insufficient surgical radicality had been subjected to radiotherapy after surgical lymph node dissection; the other 23 cases had not been subjected to radiotherapy because the aforementioned premises had been lacking. All the recurrences therefore occurred in patients with clinically and histologically ascertained metastatic lymph nodes. The presence of perilymph node metastases and the judgement of surgical radicality was thus found insufficient criteria to plan future complementary postoperative radiotherapy. However, even in those cases in which postoperative radiotherapy was performed, there was a rather high incidence of recurrences, as high as 64.7% in patients with carcinoma of the tongue. Our data indicate the opportunity of a clinical trial with preoperative radiation therapy in patients with clinically evident lymph node metastases. Thirty-six of these recurrences were situated in the upper parts of the cervical region. The prognosis is very poor in such cases, so much so that only 2 of our series were disease free at 3 years after the treatment.

Here we report 42 cases of postirradiation lesions of the brachial nerve plexus in patients treated with radiotherapy after radical mastectomy. These lesions usually appeared at least 1 year after treatment, and motorial disturbances always developed, even if initially they may have been absent. A comparison of the case material indicates that a reduction in the dose administered to the supraclavicular region and exclusion of the axillary region from the treatment resulted in a significant reduction in the incidence of these lesions. In fact, in a group of 490 patients treated by the old radiotherapy method at the Istituto Nazionale Tumori of Milan (i.e., administration of 6000 rad to the brachial nerve plexus), 16 cases of lesions at this plexus were observed. In a group of 200 patients treated instead with a new method (i.e., administration of 4900 rad to the brachial nerve plexus and exclusion of the axillary region), no nerve lesions were observed, with significance levels, according to Fisher's test, less than 0.9 %. Since radiolesions at the brachial nerve plexus have a very bad prognosis with regard to functioning of the limb and physiotherapy is of little help, this new therapeutic method is very useful, and it is not accompanied by an increase in the number of local recurrences as compared to the old method.

To investigate the relation between estrogen receptor (ER) status and timing of relapse, we retrospectively studied two groups of patients (200 cases in each group) who underwent radical mastectomy and developed an early relapse (within 3 years of the surgery) or a long-term relapse (more than 8 years after surgery). One-hundred and eighty-two (91%) patients who developed a long-term relapse were ER-positive (ER+), whereas only 64% of patients who developed an early relapse were ER+ (P<0.001), supporting the hypothesis that a long-term relapse is more frequently associated with an ER+ tumor. A review of the literature, which indicated that a long-term relapse arises more frequently in patients in whom a partial hormone control is maintained, seems to support this finding, albeit the presence of 18 ER-negative (ER-) cases in our study. However, this apparent contradictory observation could be explained by the fact that 12 of our patients were in premenopause and that ER-status could have been false ER- results due to the binding of endogenous estradiol to ER.

Jones: Orchard Therapeutics: Employment, Equity Ownership. Dott:Orchard Therapeutics: Employment, Equity Ownership. Naldini:Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI.

Genetically engineered T memory stem cells preserve differentiation activity for decades after patient infusion.

No relevant conflicts of interest to declare.

Neduva: GSK: Employment. Dow:GSK: Employment.