The comparison of dual-activity vectors with other gene delivery systems.

<p>(a) Dual-activity vectors were compared with different systems in terms of transgene expression and downregulation. (b) A549-luc-A9 cells were transfected with these vectors and after 24 h, cells were lysed. Luciferase assay was performed in order to measure the percentage of luciferase expression. (c) β-Gal expression was assessed by β-Gal assay. * p<0.05 versus naive.</p

“on and off” gene expression <i>in vitro</i> with Ad-GFP transfection and siGFP silencing.

<p>(a) Schematic representation of two-vector treatment protocols. For the two-vector approach <i>in vitro</i> cell transfections, there are three methods: pre-, co- and post- silencing. In pre-silencing, cells are treated firstly with siRNAs complexed to DOTAP:Chol liposomes and after 24 h Ads are added to the cells. In co-silencing protocol, cells are co-incubated with liposome-siRNA complexes and Ad. For post –silencing, cells are treated with liposome-siRNA complexes 24 h after Ad transfection. Cells are lysed after the last treatment in each silencing group and analyzed for gene expression. (b) C33-a cells were subjected to Ad-CMV-GFP (108 particles/mL) transfection and DOTAP:Chol liposomes complexed to siRNA (L-siGFP or L-siNeg, at 60 nM concentration) treatments at different time points as described. The cells were lysed 24 h after the last treatment and fluorescence per mg protein was measured to plot GFP expression as percentage of Ad transfected group. (c) Cell lysates were also run on SDS-PAGE electrophoresis and blotted to reveal the differences in protein levels in each treatment group. * p<0.05, ** p<0.01, *** p<0.001 versus Ad.</p

Characterization of co-enveloped Ad-siRNA vectors by DLS, TEM and AFM.

<p>The naked Ad, enveloped Ad, co-enveloped Ad-siRNA and enveloped siRNA are the vectors examined in this study. (a) The mean average diameter (nm), polydispersity index and surface charge (mV) for each vector were obtained by DLS. (b) Samples were analyzed by TEM imaging. Scale bars represent 100 nm. Red arrows indicate enveloped Ad vectors. (c) Samples were analyzed by AFM imaging.</p

The biological activity of co-enveloped Ad and siRNA in DC-Chol:DOPE.

<p>The naked Ad, DC-Chol:DOPE enveloped Ad, co-enveloped Ad-siRNA and enveloped siRNA are the vectors examined in this study. A549-luc-A9 cells were transfected with these vectors and after 24 h, cells were lysed. (a) Luciferase assay was performed in order to measure the percentage of luciferase expression. (b) β-Gal expression was assessed by β-Gal assay. * p<0.05 versus Ad.</p

Jede Stadt hat eine besondere Art, Hühner aufzuschneiden : man irrt rasch, wenn man sich auf sein Gefühl verläßt ; Christoph Antweiler fragt, was Menschsein ausmacht, was die Menschheit zusammenhält

<div><p>The ability to induce the reprogramming of somatic mammalian cells to a pluripotent state by the forced expression of specific transcription factors has helped redefine the rules of cell fate and plasticity, as well as open possibilities for disease modeling, drug screening and regenerative medicine. Here, we hypothesized that the non-viral forced expression of the four originally discovered defined factors (OKSM) in adult mice could result in <em>in vivo</em> reprogramming of cells in the transfected tissue <em>in situ</em>. We show that a single hydrodynamic tail-vein (HTV) injection of two plasmids encoding for <em>Oct3/4, Sox2, Klf4</em> and <em>c-Myc</em> respectively, are highly expressed in the liver tissue of Balb/C adult mice. Hallmark pluripotency markers were upregulated within 24–48 h after injection, followed by down-regulation of all major hepatocellular markers. Generation of transcriptionally reprogrammed cells <em>in vivo</em> was further confirmed by positive staining of liver tissue sections for all major pluripotency markers in Balb/C mice and the Nanog-GFP reporter transgenic strain (TNG-A) with concomitant upregulation of GFP expression <em>in situ</em>. No signs of physiological or anatomical abnormalities or teratoma formation were observed in the liver examined up to 120 days. These findings indicate that virus-free expression of OKSM factors <em>in vivo</em> can transcriptionally reprogram cells <em>in situ</em> rapidly, efficiently and transiently, absent of host tissue damage or teratoma formation.</p> </div

<i>In vivo</i> cell reprogramming on adult mouse liver tissue by immunohistochemistry.

<p>Balb/C mice HTV injected with 0.9% saline alone, 75 µg of pCX-OKS-2A and 75 µg pCX-cMyc in 0.9% saline, or 150 µg of pCAG-GFP in 0.9% saline and at day 4, livers were collected and frozen tissue sections were stained with anti-OCT4, anti-SOX2 or anti-NANOG antibodies to assess immunoreactivity, or BCIP/NBT to determine ALP activity in the tissue (40x). Scale bars represent 100 µm.</p

The effect of <i>in vivo</i> cell reprogramming on hepatotoxicity and liver damage.

<p>Balb/C mice HTV injected with either 75 µg of pCX-OKS-2A and 75 µg pCX-cMyc in 0.9% saline or 0.9% saline only. On days 2, 4, 8, 12, 50 and 120 livers and sera were isolated. (<b>a</b>) H&E staining of liver sections; (<b>b</b>) levels of liver enzymes and (<b>c</b>) albumin were analyzed; (<b>d</b>) liver sections were PAS stained to determine glycogen storage levels. Representative images were captured with light microscopy (10x). Scale bars represent 100 µm.</p

<i>In vivo</i> overexpression of OKSM transcription factors in adult mouse liver.

<p>Balb/C mice were HTV injected with 0.9% saline alone, 75 µg of pCX-OKS-2A and 75 µg pCX-cMyc in 0.9% saline and at days 2, 4, 8, 12, 24, RT-qPCR analysis of hepatocytes was performed to determine the relative gene expression of: (<b>a</b>) transfected transcription factors (OKSM) and (<b>b</b>) endogenous pluripotency markers. All gene expression levels were normalized to HTV-injected saline group (*p<0.05 indicates statistically significant differences between the expression levels of pluripotency markers in the OKSM and saline HTV-injected groups, obtained by the analysis of variance and Tukey's pairwise comparison); (<b>c</b>) flow cytometry analysis of OCT3/4 positive and NANOG positive cells in liver extracts; (<b>d</b>) relative gene expression of hepatocyte markers as determined by RT-qPCR. All gene expression levels were normalized to saline HTV-injected group (* p<0.05 indicates statistically significant differences between the expression levels for hepatocyte markers in the OKSM and saline HTV-injected groups, obtained by the analysis of variance and Tukey's pairwise comparison).</p

OKSM and OKS factor overexpression with dose-response in adult mouse liver.

<p>Balb/C mice HTV injected with 0.9% saline alone, pCX-OKS-2A with (OSKM) and without (OKS) pCX-cMyc in 0.9% saline, or pCAG-GFP in 0.9% saline, at the indicated doses. On day 4, RT-qPCR analysis of hepatocyte extracts was performed. (<b>a</b>) Expression levels of the injected reprogramming transcription factors and endogenous pluripotency genes were determined for plasmid dose-escalation (total plasmid dose 50, 75 and 100 µg/animal). All gene expression levels were normalized to the HTV-injected saline group (*p<0.05 indicates statistically significant differences between the expression levels of pluripotency markers in the OKSM and saline HTV-injected groups, obtained by the analysis of variance and Tukey's pairwise comparison); (<b>b</b>) Expression levels of the injected reprogramming transcription factors and endogenous pluripotency genes with and without inclusion of <i>cMyc</i>. All gene expression levels were normalized to HTV-injected saline group (**p<0.01 indicates statistically significant differences between the expression levels of pluripotency markers in the OKSM and OKS injected groups, obtained by the analysis of variance and Tukey's pairwise comparison).</p

Peptide Nanofiber Complexes with siRNA for Deep Brain Gene Silencing by Stereotactic Neurosurgery

Peptide nanofibers (PNFs) are one-dimensional assemblies of amphiphilic peptides in a cylindrical geometry. We postulated that peptide nanofibers (PNFs) can provide the tools for genetic intervention and be used for delivery of siRNA, as they can be engineered with positively charged amino acids that can electrostatically bind siRNA. The aim of this work was to investigate the use of PNFs as vectors for siRNA delivery providing effective gene knockdown. We designed a surfactant-like peptide (palmitoyl-GGGAAAKRK) able to self-assemble into PNFs and demonstrated that complexes of PNF:siRNA are uptaken intracellularly and increase the residence time of siRNA in the brain after intracranial administration. The biological activity of the complexes was investigated <i>in vitro</i> by analyzing the down-regulation of the expression of a targeted protein (BCL2), as well as induction of apoptosis, as well as <i>in vivo</i> by analyzing the relative gene expression upon stereotactic administration into a deep rat brain structure (the subthalamic nucleus). Gene expression levels of <i>BCL2</i> mRNA showed that PNF:siBCL2 constructs were able to silence the target <i>BCL2</i> in specific loci of the brain. Silencing of the <i>BCL2</i> gene resulted in ablation of neuronal cell populations, indicating that genetic interventions by PNF:siRNA complexes may lead to novel treatment strategies of CNS pathologies