Restoration of Full-Length SMN Promoted by Adenoviral Vectors Expressing RNA Antisense Oligonucleotides Embedded in U7 snRNAs

Background: Spinal Muscular Atrophy (SMA) is an autosomal recessive disease that leads to specific loss of motor neurons. It is caused by deletions or mutations of the survival of motor neuron 1 gene (SMN1). The remaining copy of the gene, SMN2, generates only low levels of the SMN protein due to a mutation in SMN2 exon 7 that leads to exon skipping. Methodology/Principal Findings: To correct SMN2 splicing, we use Adenovirus type 5–derived vectors to express SMN2antisense U7 snRNA oligonucleotides targeting the SMN intron 7/exon 8 junction. Infection of SMA type I–derived patient fibroblasts with these vectors resulted in increased levels of exon 7 inclusion, upregulating the expression of SMN to similar levels as in non–SMA control cells. Conclusions/Significance: These results show that Adenovirus type 5–derived vectors delivering U7 antisense oligonucleotides can efficiently restore full-length SMN protein and suggest that the viral vector-mediated oligonucleotide application may be a suitable therapeutic approach to counteract SMA

Numerical Investigations of Mixed Convection of Incompressible Viscous Fluid in LNG Storage with a Various Locations of Input and Output Mass

The article shows the results of mathematical simulation of mixed convection in the low-temperature storage of liquefied natural gas with a regenerative cooling. The regimes of mixed convection in a closed area with the different arrangement of the input and output sections of the masses are investigated. Two-dimensional nonstationary problem in the model of the Navier-Stokes in dimensionless variables "vorticity - stream function - temperature" was examined. Are obtained distributions of the hydrodynamic parameters and temperatures, characteristic basic laws governing the processes being investigated. Detailed circulating currents and carried out analysis of the mechanism of vortices formation and the temperature distribution in the solution for mixed convection mode with low Reynolds and Grashof numbers (Gr=10{6}, 100<Re<1000). Is established the significant influence of the geometrical arrangement of the input and output mass sections and input stream velocity on the structure of liquid flow and temperature in the low temperature LNG storage tanks

Restoration of Full-Length SMN Promoted by Adenoviral Vectors Expressing RNA Antisense Oligonucleotides Embedded in U7 snRNAs

BackgroundSpinal Muscular Atrophy (SMA) is an autosomal recessive disease that leads to specific loss of motor neurons. It is caused by deletions or mutations of the survival of motor neuron 1 gene (SMN1). The remaining copy of the gene, SMN2, generates only low levels of the SMN protein due to a mutation in SMN2 exon 7 that leads to exon skipping.Methodology/Principal FindingsTo correct SMN2 splicing, we use Adenovirus type 5–derived vectors to express SMN2-antisense U7 snRNA oligonucleotides targeting the SMN intron 7/exon 8 junction. Infection of SMA type I–derived patient fibroblasts with these vectors resulted in increased levels of exon 7 inclusion, upregulating the expression of SMN to similar levels as in non–SMA control cells.Conclusions/SignificanceThese results show that Adenovirus type 5–derived vectors delivering U7 antisense oligonucleotides can efficiently restore full-length SMN protein and suggest that the viral vector-mediated oligonucleotide application may be a suitable therapeutic approach to counteract SMA

Effect of antisense U7 snRNAs delivered by Adv-5 vectors on <i>SMN2</i> minigenes.

<p>HeLa cells were transiently transfected with either <i>SMN2</i> or <i>SMN1</i> minigenes spanning exons 6 through 8. 12 hours post transfection tissue cultures were transduced with Adv-5/antisense U7 snRNAs or control vectors (SmOpt, GFP) at a MOI of 20. The bar summarizes exon 7 inclusion levels observed from 7 independent experiments. Standard deviations are shown for each series. An asterisk above bars indicates a statistically significant difference between the experimental series and the SmOpt control series. A statistically significant difference between series is defined by p-values <0.005.</p

Evaluation of endogenous <i>SMN</i> splicing patterns in SMA type I patient-derived fibroblasts.

<p>SMA type I 3813 cells were transduced with Adv-5 vectors expressing antisense U7 snRNAs or control vectors (GFP, SmOpt) at a MOI of 20. Cells were passaged for 10 days in 2% FBS medium, and then harvested for RNA extraction. The bar graph gel image summarizes the levels observed from 8 independent experiments. Standard deviations are shown for each series. An asterisk above bars indicates a statistically significant difference between the experimental series and the SmOpt control series. A statistically significant difference between series is defined by p-values <0.005.</p

Antisense U7 snRNA strategy and selection of antisense oligonucleotides for Adenovirus delivery.

<p>(A) The modified U7 snRNA contains a sequence complementary to the 3′ splice site of <i>SMN</i> exon 8. In addition, the wild-type murine U7 Sm binding sequence was replaced with the human consensus Sm binding sequence (SmOpt) to inactivate target cleavage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008204#pone.0008204-Schumperli1" target="_blank">[15]</a>. (B) Nucleotide sequence and target location of the five different antisense oligonucleotides chosen for Adv-5 vector-derived delivery.</p

Determination of changes in functional protein levels following Adv-5/antisense U7 snRNA infection.

<p>(A) SMA type-I fibroblast cells (G3813) were transduced with Adv-5 vectors either expressing antisense U7 snRNAs or control vectors expressing SmOpt or GFP. The cell cultures were passaged for 10 days in 2% FBS medium, fixed with formaldehyde, and analyzed using immunofluorescence. As an indicator for SMN levels the fraction of nuclear gem-positive cells was determined by fluorescence microscopy. Cell nuclei are marked with white arrows. The identity of the Adv-5 vectors is indicated on the left upper corner of each panel. (B) The bar graph summarizes gem counts from 5 independent and blinded experiments. Standard deviations are shown for each series. An asterisk above bars indicates a statistically significant difference between the experimental series and the SmOpt control series. A statistically significant difference between series is defined by p-values <0.05. (C) Western blot analysis of cell cultures infected with AdV5/U7 antisense snRNPs. G3813 cells were transduced with AdV5/antisense U7 snRNPs at an MOI of 20. The expression levels were normalized to β-Actin using an anti β-Actin monoclonal antibody. Relative SMN protein levels were determined by SMN staining using a monoclonal anti SMN antibody.</p

Seamless phase IIa/IIb adaptive design with the same primary endpoint for proof of concept and dose finding

This paper considers combining a proof of concept (POC) study and a dose finding (DF) study where the POC and the DF share the same primary endpoint. An example based on real study conditions shows that compared to a conventional design the proposed adaptive design tests more active doses, with a smaller sample size and a shorter overall duration leading to a budget saving of 30% in study operations. Keywords: Adaptive design, Dose finding, Proof of concept, Seamless design, Budget savin