Optimization of short hairpin RNA for lentiviral-mediated RNAi against WAS.

International audienceThe expression of short hairpin RNAs (shRNAs) with lentiviral vectors is useful to induce stable RNA interference, particularly in hematopoietic cells. Since primary cells integrate few copies of vector, we tested if several shRNA cassette modifications could improve knock-down efficacy. Using two shRNA sequences previously shown to inhibit the human WAS gene expression, we found that neither increasing the shRNA stem length from 19-nt to 29-nt, nor modifying the loop with 4-nt, 9-nt artificial loops or with the mir30 loop improved vector-induced shRNA efficacies. This cautions against extrapolating results obtained with synthetic molecules to shRNAs that are stably expressed from viral vectors. On the other hand, the duplication of the shRNA expression cassette resulted in twice as much knock-down per copy of integrated vector. This strategy allowed a strong suppression of WASp in CD34(+) cells and will facilitate future studies on the role of WASp in human cells

Stable and functional lymphoid reconstitution in artemis-deficient mice following lentiviral artemis gene transfer into hematopoietic stem cells.

International audiencePatients with mutations in the Artemis gene display a complete absence of T- and B lymphocytes, together with increased cellular radiosensitivity; this leads to a radiosensitive severe combined immunodeficiency (RS-SCID). Allogenic hematopoietic stem-cell (HSC) transplantation is only partially successful in the absence of an human leukocyte antigen-genoidentical donor, and this has prompted a search for alternative therapeutic approaches such as gene therapy. In this study, a self-inactivated lentiviral vector expressing Artemis was used to complement the Artemis knockout mouse (Art(-/-)). Transplantation of Artemis-transduced HSCs into irradiated Art(-/-) mice restored a stable (over a 15-month period of follow-up) and functional T- and cell repertoire that was comparable to that of control mice. The success of secondary transplantations demonstrated that the HSCs had been transduced. One of thirteen mice developed a thymoma 6 months after gene therapy. Although thymic cells were seen to be carrying two lentiviral integration sites, there was no evidence of lentivirus-driven oncogene activation. The Art(-/-) mice were found to be prone to develop T-cell lymphomas, either spontaneously or after irradiation. These data indicate that the observed lymphoproliferation was probably the consequence of the chromosomal instability associated with the Artemis-deficient background. As a whole, our work provides a basis for supporting the gene therapy approach in Artemis-deficient SCID

Effects on n-3 fatty acids on depressive symptoms and dispositional optimism after myocardial infarction

Background: In patients who have experienced a myocardial infarction (MI), n-3 (omega-3) PUFA status is low, whereas the risk of depression is increased. Objective: The objective was to assess whether the plant-derived a-linolenic acid (ALA) and the fish fatty acids EPA and DHA would improve affective states. Design: In a secondary analysis of the randomized, double-blind, placebo-controlled Alpha Omega Trial, 4116 of 4837 (85.1%) patients (aged 60–80 y; 79.2% men) who had experienced an MI were included. Margarine spreads were used to deliver 400 mg EPA-DHA/d, 2 g ALA/d, both EPA-DHA and ALA, or a placebo for 40 mo. At 40 mo, the endpoints of depressive symptoms (15-item Geriatric Depression Scale) and dispositional optimism (a 4-item questionnaire and the Life Orientation Test–Revised) were analyzed by using a posttest-only design. Results: The 4 randomly assigned groups did not differ in baseline characteristics. ALA supplementation significantly increased plasma cholesteryl ester concentrations of ALA by 69%, and EPA-DHA supplementation increased plasma cholesteryl ester concentrations of EPA and DHA by 61% and 30%, respectively. Depressive symptoms or dispositional optimism did not differ between groups with the use of n-3 fatty acids compared with placebo at the 40-mo follow-up. The standardized mean (±SE) differences in depressive symptoms were as follows: for EPA-DHA plus ALA (n = 1009) compared with placebo (n = 1030), -0.025 ± 0.044 (P = 0.57); for EPA-DHA (n = 1007) compared with placebo, -0.048 ± 0.044 (P = 0.28); and for ALA (n = 1022) compared with placebo, -0.047 ± 0.044 (P = 0.29). Conclusions: In patients who had experienced an MI, low-dose EPA-DHA supplementation, ALA supplementation, or a combination of both did not affect depressive symptoms and dispositional optimism. These findings are in accord with those from previous trials in individuals without psychopathology or without severe depressive symptoms. This trial was registered at clinicaltrials.gov as NCT0012745

Outcomes following gene therapy in patients with severe Wiskott-Aldrich syndrome

International audienceIMPORTANCE: Wiskott-Aldrich syndrome is a rare primary immunodeficiency associated with severe microthrombocytopenia. Partially HLA antigen-matched allogeneic hematopoietic stem cell (HSC) transplantation is often curative but is associated with significant comorbidity. OBJECTIVE: To assess the outcomes and safety of autologous HSC gene therapy in Wiskott-Aldrich syndrome. DESIGN, SETTING, AND PARTICIPANTS: Gene-corrected autologous HSCs were infused in 7 consecutive patients with severe Wiskott-Aldrich syndrome lacking HLA antigen-matched related or unrelated HSC donors (age range, 0.8-15.5 years; mean, 7 years) following myeloablative conditioning. Patients were enrolled in France and England and treated between December 2010 and January 2014. Follow-up of patients in this intermediate analysis ranged from 9 to 42 months. INTERVENTION: A single infusion of gene-modified CD34+ cells with an advanced lentiviral vector. MAIN OUTCOMES AND MEASURES: Primary outcomes were improvement at 24 months in eczema, frequency and severity of infections, bleeding tendency, and autoimmunity and reduction in disease-related days of hospitalization. Secondary outcomes were improvement in immunological and hematological characteristics and evidence of safety through vector integration analysis. RESULTS: Six of the 7 patients were alive at the time of last follow-up (mean and median follow-up, 28 months and 27 months, respectively) and showed sustained clinical benefit. One patient died 7 months after treatment of preexisting drug-resistant herpes virus infection. Eczema and susceptibility to infections resolved in all 6 patients. Autoimmunity improved in 5 of 5 patients. No severe bleeding episodes were recorded after treatment, and at last follow-up, all 6 surviving patients were free of blood product support and thrombopoietic agonists. Hospitalization days were reduced from a median of 25 days during the 2 years before treatment to a median of 0 days during the 2 years after treatment. All 6 surviving patients exhibited high-level, stable engraftment of functionally corrected lymphoid cells. The degree of myeloid cell engraftment and of platelet reconstitution correlated with the dose of gene-corrected cells administered. No evidence of vector-related toxicity was observed clinically or by molecular analysis. CONCLUSIONS AND RELEVANCE: This study demonstrated the feasibility of the use of gene therapy in patients with Wiskott-Aldrich syndrome. Controlled trials with larger numbers of patients are necessary to assess long-term outcomes and safety