Gene therapy has been proposed as a potential treatment for Wiskott-Aldrich syndrome (WAS), a severe primary immune deficiency characterized by multiple hematopoietic-specific cellular defects. In order to develop an optimal lentiviral gene transfer cassette for this application, we compared the performance of several internal promoters in a variety of cell lineages from human WAS patients. Vectors using endogenous promoters derived from short (0.5 kb) or long (1.6 kb) 5' flanking sequences of the WAS gene, expressed the transgene in T, B, dendritic cells as well as CD34(+) progenitor cells, but functioned poorly in non-hematopoietic cells. Defects of T-cell proliferation and interleukin-2 production, and the cytoskeletal anomalies in WAS dendritic cells were also corrected. The levels of reconstitution were comparable to those obtained following transduction with similar lentiviral vectors incorporating constitutive PGK-1, EF1-alpha promoters or the spleen focus forming virus gammaretroviral LTR. Thus, native regulatory sequences target the expression of the therapeutic WAS transgene to the hematopoietic system, as is naturally the case for WAS, and are effective for correction of multiple cellular defects. These vectors may have significant advantages for clinical application in terms of natural gene regulation, and reduction in the potential for adverse mutagenic events.

Adeno-associated virus (AAV) vectors are a platform of choice for in vivo gene transfer applications. However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration. In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration. Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples. The assay is easy to setup and sensitive and, depending on the purpose, can be validated to support clinical development of gene therapy products based on AAV vectors.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked neuromuscular disease that affects 1 boy in 3500 to 5000 boys. The golden retriever muscular dystrophy dog is the best clinically relevant DMD animal model. Here, we used a high-thoughput miRNA sequencing screening for identification of candidate serum miRNA biomarkers in golden retriever muscular dystrophy dogs. We confirmed the dysregulation of the previously described muscle miRNAs, miR-1, miR-133, miR-206, and miR-378, and identified a new candidate muscle miRNA, miR-95. We identified two other classes of dysregulated serum miRNAs in muscular dystrophy: miRNAs belonging to the largest known miRNA cluster that resides in the imprinting DLK1-DIO3 genomic region and miRNAs associated with cardiac disease, including miR-208a, miR-208b, and miR-499. No simple correlation was identified between serum levels of cardiac miRNAs and cardiac functional parameters in golden retriever muscular dystrophy dogs. Finally, we confirmed a dysregulation of miR-95, miR-208a, miR-208b, miR-499, and miR-539 in a small cohort of DMD patients. Given the interspecies conservation of miRNAs and preliminary data in DMD patients, these newly identified dysregulated miRNAs are strong candidate biomarkers for DMD patients.

The development of medical approaches requires preclinical and clinical trials for assessment of therapeutic efficacy. Such evaluation entails the use of biomarkers, which provide information on the response to the therapeutic intervention. One newly-proposed class of biomarkers is the microRNA (miRNA) molecules. In muscular dystrophies (MD), the dysregulation of miRNAs was initially observed in muscle biopsy and later extended to plasma samples, suggesting that they may be of interest as biomarkers. First, we demonstrated that dystromiRs dysregulation occurs in MD with either preserved or disrupted expression of the dystrophin-associated glycoprotein complex, supporting the utilization of dystromiRs as generic biomarkers in MD. Then, we aimed at evaluation of the capacity of miRNAs as monitoring biomarkers for experimental therapeutic approach in MD. To this end, we took advantage of our previously characterized gene therapy approach in a mouse model for α-sarcoglycanopathy. We identified a dose-response correlation between the expression of miRNAs on both muscle tissue and blood serum and the therapeutic benefit as evaluated by a set of new and classically-used evaluation methods. This study supports the utility of profiling circulating miRNAs for the evaluation of therapeutic outcome in medical approaches for MD.

The Wiskott Aldrich syndrome protein (WASP) is a hematopoietic-specific cytoskeletal regulator that is necessary for induction of normal immunity. In the context of effective gene therapy for WAS, cellular models of human WASP deficiency are important for definition of the threshold of protein expression required for optimal activity. Using lentiviral vector-mediated RNA interference (RNAi), we were able to down-regulate the levels of human WASP in cell lines and primary cells. In dendritic cells (DC), RNAi-induced WASP deficiency did not impair phenotypic maturation but perturbed cytoskeletal organization. As a result, podosomes, which are actin-rich structures present in immature adherent DC, were formed less efficiently and motility was disturbed. Overall, treatment of cells with RNAi recapitulated the phenotype of cells derived from patients or animals with inactivating mutations of the WAS gene. Interestingly, reduction of the levels of WASP to about 60% of normal was sufficient to inhibit the formation of podosomes in DC, implying that this cell type requires near-normal levels of WASP to sustain physiological cytoskeleton-dependent activities.

Motivation: Piwi-interacting RNA (piRNA) is the most recently discovered and the least investigated class of Argonaute/Piwi protein-interacting small non-coding RNAs. The piRNAs are mostly known to be involved in protecting the genome from invasive transposable elements. But recent discoveries suggest their involvement in the pathophysiology of diseases, such as cancer. Their identification is therefore an important task, and computational methods are needed. However, the lack of conserved piRNA sequences and structural elements makes this identification challenging and difficult.Results: In the present study, we propose a new modular and extensible machine learning method based on multiple kernels and a support vector machine (SVM) classifier for piRNA identification. Very few piRNA features are known to date. The use of a multiple kernels approach allows editing, adding or removing piRNA features that can be heterogeneous in a modular manner according to their relevance in a given species. Our algorithm is based on a combination of the previously identified features [sequence features (k-mer motifs and a uridine at the first position) and piRNAs cluster feature] and a new telomere/centromere vicinity feature. These features are heterogeneous, and the kernels allow to unify their representation. The proposed algorithm, named piRPred, gives promising results on Drosophila and Human data and outscores previously published piRNA identification algorithms.Availability and implementation: piRPred is freely available to non-commercial users on our Web server EvryRNA http://EvryRNA.ibisc.univ-evry.frContact: tahi@ibisc.univ-evry.fr

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The heterodimer Ku70/80 Ku is the DNA-binding component of the DNA-PK complex required for the nonhomologous end-joining pathway. It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication, and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-long terminal repeat (LTR) viral DNA circles, viral DNA integration, and transcription. We describe here the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analyzed the transgene distributions localization in nucleus by applying a three-dimensional reconstruction model to two-dimensional fluorescence in situ hybridization images. This indicated that the presence of Ku80 resulted in a bias toward the transgenes being located at the periphery of the nucleus associated with their being repressed; in the absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.