A CMV-H1 hybrid construct driving shLuc provides enhanced inhibition of a co-transfected target gene in the newborn (a and b) and in the adult (c) mouse brain

<p><b>Copyright information:</b></p><p>Taken from "A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain"</p><p></p><p>Nucleic Acids Research 2007;35(9):e65-e65.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1888798.</p><p>© 2007 The Author(s)</p> () Dose dependence of CMV-H1-shLuc efficiency. The inhibition efficiency of CMV-H1-shLuc was tested at different doses ranging from 0.1 to 0.4 µg/µl. After co-transfection of pGL2-CMV and pRL-CMV along with 0.4 µg/µl of CMV-H1-shLuc (i.e. 0.8 µg/hemisphere), we observed up to 50% inhibition of the targeted luciferase expression. This level of inhibition was obtained at 48 h post-transfection. () Time course efficiency of CMV-H1-shLuc. Significant inhibition of the target gene with 0.4 µg/µl of CMV-H1-shLuc was seen at all times tested. The maximal level of inhibition (50%) was seen at 50 h post-transfection. () In the adult brain, H1-shLuc provided no inhibition of PP:RL ratio (grey bars) compared to controls (black bars). CMV-H1-shLuc leads to 25% inhibition of the target gene at 72 h post-transfection (white bars) and up to 112 h post-transfection (data not shown). Means ± SEM are shown. NS = ‘not significant’; * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001.  = 10 injected hemispheres per group

Sequences of the oligopyrimidine•oligopurine DNA targets () and of the oligonucleotides () used in this study

<p><b>Copyright information:</b></p><p>Taken from "Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities"</p><p>Nucleic Acids Research 2005;33(13):4223-4234.</p><p>Published online 27 Jul 2005</p><p>PMCID:PMC1181241.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> (A) The wild-type PPT target sequence (boxed), Dra I recognition site (underlined) are shown, as well as the mutated PPT duplex presenting two mutations (in bold). (B) (T,C)-containing TFOs directed against the wild-type PPT duplex and control sequences. Small letters indicate LNA nucleotides and capitals DNA nucleotides. Cytosines in italic ( and ) are methylated at position 5; all cytosines (LNA and DNA) are 5-methylated. The abbreviated names are indicated near the sequences. (po) stands for phosphodiester. Last four TFOs are either acridine (Acr) or psoralen (Pso) 5′-conjugated TFO/LNAs

Quantification of topo II-mediated DNA cleavage in the presence of TFO–drug conjugates

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides"</p><p>Nucleic Acids Research 2006;34(6):1900-1911.</p><p>Published online 5 Apr 2006</p><p>PMCID:PMC1447649.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The analysis was as in and the gels were quantified after normalization relative to total radioactivity loaded. () Scheme showing the enhanced cleavage site of each conjugate: the 5′ conjugates are depicted in green as the corresponding cleavage site a; the 3′ 16TFO conjugates are depicted in red as the corresponding cleavage site f; the 3′ 20TFO conjugates are depicted in blue as the corresponding cleavage site g. The other topo II-mediated DNA cleavage sites described in the text are also labeled with letters. () Quantification of the specific cleavage for 20TFO-L- on both strands compared to free 20TFO-L. The cleavage intensity was normalized to the cleavage intensity of the free drug (at 1 µM) on a logarithmic scale at each cleavage site. The oligopyrimidine strand is in gray and the oligopurine strand is in black, the conjugate in filled bars and the 20TFO-L alone in hatched bars. () Specific cleavage intensities of the conjugates on the oligopyrimidine-containing strand of the duplex (Y). The 5′ 16TFO conjugates are depicted in light green (hatched bars -L-16TFO, squares -L-16TFO, horizontal bars -L-16TFO, vertical bars -L-16TFO), the 5′ 20TFO conjugates are depicted in dark green (hatched bars -L-20TFO, vertical bars -L-20TFO), the 3′ 16TFO conjugates are in red [hatched bars 16TFO-L-, squares 16TFO-L-, horizontal bars 16TFO-L-, vertical bars 16TFO-L-, crosses 16TFO-L-(4)] and the 3′ 20TFO ones are in blue [hatched bars 20TFO-L-, vertical bars 20TFO-L-, crosses 20TFO-L-(4), dots 20TFO-L-(4)]

The sequence of the target duplex and the TFOs, and the chemical structure of the drug–TFO conjugates

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides"</p><p>Nucleic Acids Research 2006;34(6):1900-1911.</p><p>Published online 5 Apr 2006</p><p>PMCID:PMC1447649.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The 77 bp duplex target sequence was inserted between the BamHI and EcoRI sites of pBSK. The TFO is complementary to the oligopurine strand of the duplex and binds parallel to it. The target site is in boldface for the 20 nt TFO and is underlined for the 16 nt TFOs. , 5-methyl-2′-deoxycytidine; , 5-propynyl-2′-deoxyuridine. The structures of the VP16 derivatives-TFO conjugates used in this study are shown. The nomenclature of the conjugates is described in the Materials and Methods

Structural remodelling and altered diastolic function in hearts of <i>Dmd^mdx</i> rats.

<p>Echocardiography was performed on 3 month-old <i>Dmd^mdx</i> rats. The structural remodeling and the systolic and diastolic functions were assessed respectively by Two-dimensional (2-D) echocardiography and pulsed Doppler (n = 6 for each condition); Values are mean ± SEM; *p<0.05). (TM mode: Time movement mode; LV: Left ventricular; E/A ratio: early diastolic (E)/late diastolic (A) ratio).</p

Rat weight and size as well as isolated muscles weight and heart characteristics of <i>Dmd^mdx</i> male rats.

<p>body mass index calculated as body weight/(body length)²; (g/cm²).</p><p>Values are means ± SEM; *: p<0.05 <i>vs</i> wild-type controls (WT); §: p<0.0001 <i>vs</i> WT. n, numbers of rats.</p><p>Rat weight and size as well as isolated muscles weight and heart characteristics of <i>Dmd^mdx</i> male rats.</p

Severe and progressive muscle changes in <i>Dmd^mdx</i> rats.

<p><i>Biceps femoris</i> muscle was sampled at 3 and 7 month-old from wild-type littermate controls (WT) and <i>Dmd^mdx</i> rats. Compared to controls (<i>A</i>), 3 month-old <i>Dmd^mdx</i> rat skeletal muscles (<i>B</i>) displayed individual fiber necrosis (open arrowhead) associated with foci of small newly regenerating centronucleated fibers (arrow). In addition, 7 month-old muscles displayed a progessive replacement of fibers by fibrosis (*) and fat (black arrowhead) tissue (<i>C</i>). The regenerative activity was assessed using a specific antibody against the Myosin Heavy Chain developmental isoform (MyoHC_Dev). No regenerative activity was observed in control rats (<i>D</i>) whereas newly regenerating fibers were numerous in <i>Dmd^mdx</i> rats (<i>E</i>) with a decrease in their number with age (<i>F</i>). Histological changes were quantified. Fiber minimal Ferret diameter was measured. A global switch towards lower sized fibers was observed in the fiber size distribution in mutated rats compared to controls at 3 and 7 month of age (<i>G–H</i>). Hemalun eosin saffron staining (<i>A–C</i>), immunolabelling of Myosin Heavy Chain developmental isoform (MyoHC_Dev) (<i>D–F</i>). Bar = 100 µm (<i>A–C</i>) and 200 µm (<i>D–F</i>).</p

<i>Dmd^mdx</i> rats are characterized by muscle weakness and by a decrease in a spontaneous activity.

<p>Forelimb grip test and locomotor activity of <i>Dmd^mdx</i> rats were analyzed at the age of 3 months. Compared to wild-type littermate controls (WT), <i>Dmd^mdx</i> rats were characterized by muscle weakness and showed a lower strength in absolute values (<i>A</i>) and when normalized to body weight (<i>B</i>). <i>Dmd^mdx</i> rats showed fatigue with a decreased force grip across the five pulls, and significant values are obtained for the two last trials that were reduced to ∼70% when compared to Trial 1. At 3 months, when compared to WT, <i>Dmd^mdx</i> rats were less active and the number of movements (<i>C</i>), the total distance travelled (<i>C</i>), the time of activity (<i>D</i>) and the number of rearing (<i>D</i>) were significantly lower. Values are means ± SEM; *p<0.05 <i>vs</i> WT; §: p<0.05 <i>vs</i> Trial 1.</p

No dystrophin expression was detected in cardiac and skeletal muscles of <i>Dmd^mdx</i> rats.

<p>(<i>A</i>) Male 7 month-old rats of line 61, wild-type littermate controls (WT) and <i>Dmd^mdx</i> were sacrificed and biopsies from <i>tibialis cranialis</i> muscles (TC) and hearts (H) were harvested. Western-blot of total proteins (50 µg) was incubated with NCL-DYS2 and Manex1011C monoclonal antibodies (C-terminal and exons 10/11 epitopes, respectively). This revealed undetectable levels of the 427 kDa dystrophin band in line 61 <i>Dmd^mdx</i> rats. Muscle from a GRMD dog (GD) was used as negative control and samples from WT rats were used as positive controls. Staining with an anti-GAPDH polyclonal antibody validated equal protein loadings. (<i>B–E</i>) Heart and <i>biceps femoris</i> muscles from the same wild-type (<i>B</i> and <i>C</i>) and <i>Dmd^mdx</i> rats (<i>D</i> and <i>E</i>) were assessed for dystrophin expression using immunohistochemistry with Mandys110 monoclonal antibody (against exons 38–39 epitope). Compared to the subsarcolemmal expression of dystrophin in wild-type muscles, no dystrophin was detected in <i>Dmd^mdx</i> rats except for the presence in skeletal muscle of only rare scattered revertant positive fibers (arrowheads). Immunolabelling of dystrophin (<i>B–E</i>) Bar = 100 µm.</p

Progressive myofiber replacement by fibrotic and fat tissue in <i>Dmd^mdx</i> rats.

<p>A picrosirius red staining specific for fibrosis was performed on <i>biceps femoris (A–C)</i>, respiratory (<i>D–F</i>) and heart muscle (<i>G–I</i>) samples obtained from wild-type littermate controls (WT) and 3 month-old and 7 month-old <i>Dmd^mdx</i> rats. Compared to control rats (left panel), a progressive increase in the amount of fibrotic tissue (black arrowhead) was noticed in 3 (mid panel) and 7 month-old <i>Dmd^mdx</i> rats (right panel). Note the focal presence of fat tissue infiltration (open arrowhead). In the heart, fibrosis was most marked in papillary muscle of the left ventricle (LV), in the septum and in the ventricular subepicardic area. Picrosirius red staining. Bar = 100 µm (A<i>–</i>F) and 1 mm (G<i>–</i>I).</p