Hypomorphic RAG deficiency: impact of disease burden on survival and thymic recovery argues for early diagnosis and HSCT.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.

Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Pre-transplant donor CD4- invariant NKT cell expansion capacity predicts the occurrence of acute graft-versus-host disease.

Clinically useful pre-transplant predictive factors of acute graft-versus-host-disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT) are lacking. We prospectively analyzed HSC graft content in CD34+, NK, conventional T, regulatory T and invariant natural killer T (iNKT) cells in 117 adult patients before allo-SCT. Results were correlated with occurrence of aGVHD and relapse. In univariate analysis, iNKT cells were the only graft cell populations associated with occurrence of aGVHD. In multivariate analysis, CD4- iNKT/T cell frequency could predict grade II-IV aGVHD in bone marrow and peripheral blood stem cell (PBSC) grafts, while CD4- iNKT expansion capacity was predictive in PBSC grafts. Receiver operating characteristic analyses determined the CD4- iNKT expansion factor as the best predictive factor of aGVHD. Incidence of grade II-IV aGVHD was reduced in patients receiving a graft with an expansion factor above versus below 6.83 (9.7 vs 80%, P<0.0001), while relapse incidence at two years was similar (P=0.5).The test reached 94% sensitivity and 100% specificity in the subgroup of patients transplanted with human leukocyte antigen 10/10 PBSCs without active disease. Analysis of this CD4- iNKT expansion capacity test may represent the first diagnostic tool allowing selection of the best donor to avoid severe aGVHD with preserved graft-versus-leukemia effect after peripheral blood allo-SCT.

Lymphopoiesis in transgenic mice over-expressing Artemis.

Artemis is a factor of the non-homologous end joining pathway involved in DNA double-strand break repair that has a critical role in V(D)J recombination. Mutations in DCLRE1C/ARTEMIS gene result in radiosensitive severe combined immunodeficiency in humans owing to a lack of mature T and B cells. Given the known drawbacks of allogeneic hematopoietic stem cell transplantation (HSCT), gene therapy appears as a promising alternative for these patients. However, the safety of an unregulated expression of Artemis has to be established. We developed a transgenic mouse model expressing human Artemis under the control of the strong CMV early enhancer/chicken beta actin promoter through knock-in at the ROSA26 locus to analyze this issue. Transgenic mice present a normal development, maturation and function of T and B cells with no signs of lymphopoietic malignancies for up to 15 months. These results suggest that the over-expression of Artemis in mice (up to 40 times) has no deleterious effects in early and mature lymphoid cells and support the safety of gene therapy as a possible curative treatment for Artemis-deficient patients.

AK2 deficiency compromises the mitochondrial energy metabolism required for differentiation of human neutrophil and lymphoid lineages.

Reticular dysgenesis is a human severe combined immunodeficiency that is primarily characterized by profound neutropenia and lymphopenia. The condition is caused by mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of mitochondrial AK2 protein expression. AK2 regulates the homeostasis of mitochondrial adenine nucleotides (ADP, ATP and AMP) by catalyzing the transfer of high-energy phosphate. Our present results demonstrate that AK2-knocked-down progenitor cells have poor proliferative and survival capacities and are blocked in their differentiation toward lymphoid and granulocyte lineages. We also observed that AK2 deficiency impaired mitochondrial function in general and oxidative phosphorylation in particular - showing that AK2 is critical in the control of energy metabolism. Loss of AK2 disrupts this regulation and leads to a profound block in lymphoid and myeloid cell differentiation.

Gene therapy for primary immunodeficiencies.

Gene therapy has effectively entered Medicine via the field of primary immunodeficiencies (PID). Because hematopoietic stem cells are accessible and because it was understood that genetic correction of lymphocyte progenitor cells carrying a genetic defect impairing differentiation, could result in the production of long-lived T lymphocytes, it was reasoned that ex vivo gene transfer in hematopoietic cells could lead to disease phenotype correction. Retroviral vectors were designed to ex vivo transduce such cells. This has indeed been shown to lead to sustained correction of the T cell immunodeficiency associated with two forms of severe combined immunodeficiencies (SCID) for now more than ten years. Occurrence in some patients of genotoxicity related to retroviral vectors integration close to and transactivation of oncogenes has led to the development of retroviral vectors devoid of its enhancer element. Results of recent trials performed for several forms of PID indeed suggest that their use is both safe and efficacious. It is thus anticipated that their application to the treatment of many more life threatening PID will be developed over the coming years.

Variable correction of Artemis deficiency by I-Sce1-meganuclease-assisted homologous recombination in murine hematopoietic stem cells.

The correction of genetic mutations by homologous recombination is an attractive approach to gene therapy. We used the DNA double-strand breaks introduced by the site-specific endonuclease I-Sce1 as a means of increasing homologous recombination of an exogenous DNA template in murine hematopoietic stem cells (mHSCs). To develop this approach, we chose an Artemis knockout (Art(-/-)) mouse in which exon 12 of the Artemis gene had been replaced by an I-Sce1 recognition site. The I-Sce1 enzyme and the Artemis correction template were each delivered by a self-inactivating (SIN)-integrase-defective lentiviral vector (SIN-IDLV-CMV-ISce1 and SIN-IDLV-Art, respectively). Transduction of Art(-/-) mHSCs with the two vectors successfully reverted the Art(-/-) phenotype in 2 of our 10 experiments. Even though the potential for genotoxicity has yet to be evaluated, this new approach to gene editing appears to be promising. Improving the efficacy of this approach will require further technical work.

Safety of hematopoietic stem cell transplantation from hepatitis B core antibodies-positive donors with low/undetectable viremia in HBV-naïve children.

Scarce data exist on allogeneic hematopoietic stem cell transplantation (HSCT) outcomes in hepatitis B virus (HBV)-naïve recipients from HBV-experienced donors. Long-term follow-up is herein reported for 17 allogeneic HSCT performed in 13 HBV-naïve children from HBc-antibodies-positive donors between 2006 and 2012. Four donors were HBs-antigen-positive, with detectable but low viremia in 2 cases (<2 log10IU/ml). HBV-DNA was undetectable in all transplanted cell products. Recipients' HBV prophylaxis consisted of pre-transplant vaccination, polyvalent immune globulins, specific anti-HBV immune globulins, and/or oral lamivudine in 3, 12, 8, and 8 children, respectively. No case of HBV transmission occurred based on negative close monitoring of recipients' HBV serology and plasma HBV-DNA during a median follow-up of 22 months. In case of undetectable viremia in the donor, prophylaxis with vaccination and/or immune globulins in the recipient seems to be sufficient and lamivudine prophylaxis might be unnecessary to prevent viral transmission. In case of undetectable viremia in the donor, a systematic screening of HBV DNA in the stem cell product might be unnecessary to confirm the low risk of viral transmission. Prior exposure to HBV in the donor should not be considered a contraindication to HSCT.

Use of liquid culture and cell cycle analysis to compare drug damage following in vitro treatment of normal human bone marrow cells with adriamycin, arabinosyl-cytosine, and etoposide.

The effects of adriamycin (ADM), arabinosyl-cytosine (ARA-C), and etoposide (VP16) were studied on human bone marrow mononucleated cells using colony formation in agar, a modified liquid culture system, and flow cytometry analysis of the cell cycle. Drug concentrations tested during a 1-h incubation ranged from 0.1 to 4 micrograms/ml for ADM, from 0.3 to 30 micrograms/ml for VP16, and from 10(-7) to 10(-3) M for ARA-C. Regression analysis of the dose-response curves was used to assess the drug concentration that inhibited 90% +/- 5% (LD90) of colony growth. LD90s were 0.4 microgram/ml for ADM, 20 micrograms/ml for VP16, and 10(-4) M for ARA-C. LD90-surviving cells were cultured in liquid medium for 3 weeks. Surviving cells over this time were 13% of the control for ADM, 22% for VP16, and 95.7% for ARA-C. Although cells decreased drastically in ADM- and VP16-treated samples, granulocyte-macrophage colony-forming units (CFU-GM) per 10(5) surviving cells rose to twice the control for ADM, to 60% for VP16, and to 150% for ARA-C. Flow cytometry analysis of the cell cycle was performed at day 0 and at day 4 after treatment with the LD90 dose. It showed a rapid and reversible effect of ARA-C on cells in the S-phase, whereas the action of VP16 concerned all cells, regardless of their cycle phase. We conclude that the direct effects of the three drugs on CFU-GM in agar are poorly predictive of hematopoietic reconstitution capacity, except for VP16. Liquid culture gives a much more accurate appraisal of the long-term damage and recovery due to anticancer drugs.

[Prevention of bacterial infections after bone marrow graft by broad-spectrum oral antibiotics, absorbable (pefloxacin, penicillin) and non absorbable (cephalosporin, gentamycin, bacitracin)]. / Prévention des infections bactériennes après greffe de moelle osseuse par antibiotiques oraux à large spectre absorbables (pefloxacine, pénicilline) ou non absorbables (céphalosporine, gentamicine, bacitracine).

In a consecutive series of 65 patients treated by allogeneic bone marrow transplantation, we have compared the efficacy on prevention of bacterial infection of total gut decontamination with oral non absorbable antibiotics or with oral absorbable broad spectrum antibiotics. On day-8, all patients were randomly allocated to one group: Group I received orally pefloxacin 400 mg/day and penicillin 3 M UI/day. Group II received capsules containing cephalothin 250 mg, gentamicin 20 mg and bacitracin 150 UI. Each patient received 9 to 12 capsules per day according to body weight. Patients less than 5 years old or with severe hepatic abnormalities were excluded from the study. All patients were treated in LAF room with usual precautions of asepsis. They received sterile food. In addition, they were on ketoconazole for prophylaxis of fungal infections and acyclovir for prevention of herpes infections. Antibiotics were started on day-8 before bone marrow transplantation and stopped 15 days after discharge from the hospital. 32 patients were allocated to Group I and 33 in Group II. There was no difference between both groups according to age, sex, diagnosis. After transplant, the one year survival and the complications were similar in both groups. The median time of decontamination was 65 days, the mean number of days of agranulocytosis was 20 days, the mean number of days of fever was 7 days in both groups. The compliance was better in Group I but the treatment had to be modified in 9 patients of Group I because of liver abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

[Prevention and treatment of cytomegalovirus infections after graft of allogenic bone marrow]. / Prévention et traitement des infections à cytomégalovirus après greffe de moelle osseuse allogénique.

Cytomegalovirus (CMV) infection is the most frequent cause of lethal infection after bone marrow transplantation. Viremia occurs in 50% of patients seropositive for CMV before transplantation. Interstitial pneumonitis due to CMV occurs in 10% to 20% of patients with 85% mortality. It is known that CMV infection is due to host reactivation of latent CMV infection or to the transmission of the virus by the marrow donor or by blood transfusions. Treatment of CMV infection has been disappointing in the past. All attempts to treat CMV pneumonia with available agents have failed. Recent studies have indicated the usefulness of prophylactic measures and the early treatment of CMV infections. The use of hyperimmune gammaglobulins has given contradictory results. The selection of seronegative marrow donors or blood donors is useful only if the recipient is seronegative. New antiviral drugs have been used recently in preliminary clinical trials. In preliminary studies a guanosine analogue similar to Acyclovir (DHPG Synthex or BWB 759 U Wellcome) has given reasonable hope of disease cure if it is used early before the occurrence of pneumonia. Phosphonoformate (Foscarnet) has also been shown to be active against CMV infection. Both drugs have good antiviral and clinical action in immunosuppressed patients but the results have been disappointing in cases of pneumonia. Relapse occurs frequently after cessation of the treatment and attempts are being made to use maintenance therapy.