Bioluminescence imaging of gene transduction by vectors derived from AAV2, -8 and -9 displaying peptide ligands.

<p>In vivo bioluminescence imaging of transgene expression in FVB mice injected intravenously with rAAV-luciferase vectors harboring wild type capsid or capsids displaying a targeting peptide selected in the structural context of AAV2. Images were taken 28 days after vector injection, when whole animal bioluminescence intensities (BLI) reached peak values after injection of luciferase substrate. BLI ranges from 10⁵–10⁸ relative light units per animal (photons/sec/cm²).</p

In vivo bioluminescence of gene expression in breast cancer tissue of PymT-transgenic mice.

<p>Luciferase vectors derived from AAV2, -8 and -9 displaying breast cancer-directed peptides (ESGLSQS) and respective wild type capsid vectors were injected intravenously into tumor-bearing mice (n = 3 per group). Images were taken 14 days after vector injection. BLI ranged from 10⁵–10⁸ relative light units per animal (photons/sec/cm²).</p

Biodistribution analysis of tropism-modified vectors.

<p>Vector genome copy numbers in various tissues were quantified by quantitative real-time PCR using CMV-promoter-specific primers. Tissues were harvested 28 days after intravenous injection of capsid-modified or wild type vectors, respectively. Recovered vector genome numbers are shown for <b>A</b> liver, <b>B</b> cardiac, and <b>C</b> lung tissue. (n = 3 animals per group).</p

Titers of recombinant AAV vectors.

<p>*vector genomes of viral stocks were determined by quantitative real-time PCR.</p

Ex vivo determination of transgene expression after systemic administration of AAV2, AAV8 and AAV9 vectors displaying peptide ligands.

<p>Luciferase gene expression in various tissues was analyzed 28 days after intravenous injection of rAAV vectors into FVB mice. Capsid insertion of the random VRRPRFW peptide did not result in transgene expression in any organ, while the lung-directed peptide PRSTSDP and the breast tissue-directed peptide ESGLSQS resulted in specific changes of tissue tropism. <b>A</b> shows results for AAV2-, <b>B</b> for AAV8-, and <b>C</b> for AAV9-injected animals. Values below 10³ RLU/mg protein are not shown because this indicates the threshold beyond which luciferase expression data could be reproducibly delineated from background signal. * = p<0.05; ** = p<0.01; *** = p<0.001 capsid displaying peptide versus respective wild type control (n = 3 animals).</p

Breast cancer-directed gene delivery by AAV capsids displaying ESGLSQS depends on the underlying capsid serotype.

<p><b>A:</b> Luciferase activities in PymT-induced breast cancer tissue were determined as relative light units (RLU) per mg protein 14 days after injection of AAV2, -8 and -9 displaying breast cancer-directed peptides (ESGLSQS) or respective wild type capsid variants (n = 3). <b>B and C:</b> the amount of vector genomes recovered in tumor, liver and cardiac tissue was analyzed by quantitative real-time PCR using the tissue samples as described in A (n = 3 animals per group). <b>B:</b> ratio of vector genome copy numbers tumor/liver (n = 3 animals per group); <b>C:</b> ratio of vector genome copy numbers tumor/heart. <b>D and E:</b> ratios of gene transduction using AAV vectors of serotypes 2, 8 and 9 with insertion of the ESGLSQS peptide and respective wild type variants. Values were calculated as <b>D:</b> tumor/liver expression ratios or <b>E:</b> tumor/heart expression ratios, respectively. <b>F, G and H:</b> Luciferase activities in various tissues were detected as relative light units (RLU) per mg protein for AAV-2 (<b>F</b>) for AAV-8 (<b>G</b>) and AAV-9 (<b>H</b>) displaying breast cancer-directed peptides (ESGLSQS) or respective AAV wild type capsid variants (n = 3 animals per group).</p

Design of novel peptide insertion sites in capsid regions adjacent to the threefold-spike in serotypes AAV8 and AAV9.

<p><b>A:</b> Sequence alignment of surface exposed capsid domains encoded by the <i>cap</i> gene of the AAV serotypes 2, 8, and 9. Parts of the heparin binding domain 484-RQQR-487 and the integrin binding motif 511-NGR-513 are strongly conserved among the three serotypes. While AAV8 and -9 both contain an R at position 532, marked differences in the sequences are apparent at positions 585–588. Domains highlighted in B are tagged by colored rectangles. Alignment of the serotype sequences was carried out using BioEdit Sequence Alignment Editor Software. <b>B:</b> Capsid domains of AAV2 known to be involved in receptor binding are highlighted in blue (R484; R487), yellow (511-NGR-513), green (K532), and red (R585; R588). Surface rendering and mapping of the threefold-spike region was performed using PyMOL with the crystal structure of AAV2 supplied as template (PDB ID: 1lp3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023101#pone.0023101-Xie1" target="_blank">[50]</a>). <b>C:</b> Design of AAV8 and -9 constructs for the incorporation of oligonucleotides encoding targeting peptides into the <i>cap</i> gene. The amino acid sequence of the <i>cap</i> gene for each corresponding serotype (single letter amino acid code) is depicted in black letters; differences compared to the respective wild type sequence are shown in blue letters. Red indicates seven additional amino acid residues from the insertion of oligonucleotides encoding a known peptide ligand for the re-direction of AAV serotypes. <b>D:</b> Model of the VP-3 capsid protein of AAV2 and localization of R588 and <b>E:</b> model of the VP-3 protein of AAV8 capsid and localization of R590. The potential peptide insertion site is depicted in yellow. Basic amino acids are depicted in red, acidic amino acids in blue. The VP-3 model of AAV2 and AAV8 was generated using Cn3D with coordinates PDB ID: 1lp3 for AAV2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023101#pone.0023101-Xie1" target="_blank">[50]</a> and PDB ID: 2QA0 for AAV8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023101#pone.0023101-Nam1" target="_blank">[37]</a> serving as template.</p

Effects of Increased Von Willebrand Factor Levels on Primary Hemostasis in Thrombocytopenic Patients with Liver Cirrhosis

<div><p>In patients with liver cirrhosis procoagulant and anticoagulant changes occur simultaneously. During primary hemostasis, platelets adhere to subendothelial structures, via von Willebrand factor (vWF). We aimed to investigate the influence of vWF on primary hemostasis in patients with liver cirrhosis. Therefore we assessed in-vitro bleeding time as marker of primary hemostasis in cirrhotic patients, measuring the Platelet Function Analyzer (PFA-100) closure times with collagen and epinephrine (Col-Epi, upper limit of normal ≤165 s) or collagen and ADP (Col-ADP, upper limit of normal ≤118 s). If Col-Epi and Col-ADP were prolonged, the PFA-100 was considered to be pathological. Effects of vWF on primary hemostasis in thrombocytopenic patients were analyzed and plasma vWF levels were modified by adding recombinant vWF or anti-vWF antibody. Of the 72 included cirrhotic patients, 32 (44.4%) showed a pathological result for the PFA-100. They had mean closure times (± SD) of 180±62 s with Col-Epi and 160±70 s with Col-ADP. Multivariate analysis revealed that hematocrit (<i>P</i> = 0.027) and vWF-antigen levels (<i>P</i> = 0.010) are the predictors of a pathological PFA-100 test in cirrhotic patients. In 21.4% of cirrhotic patients with platelet count ≥150/nL and hematocrit ≥27.0%, pathological PFA-100 results were found. In thrombocytopenic (<150/nL) patients with cirrhosis, normal PFA-100 results were associated with higher vWF-antigen levels (462.3±235.9% vs. 338.7±151.6%, <i>P</i> = 0.021). These results were confirmed by multivariate analysis in these patients as well as by adding recombinant vWF or polyclonal anti-vWF antibody that significantly shortened or prolonged closure times, respectively. In conclusion, primary hemostasis is impaired in cirrhotic patients. The effect of reduced platelet count in cirrhotic patients can at least be partly compensated by increased vWF levels. Recombinant vWF could be an alternative to platelet transfusions in the future.</p></div

Effect of von Willebrand factor on primary hemostasis in thrombocytopenic patients with liver cirrhosis.

<p>Increased levels of vWF were associated with maintained normal primary hemostasis in patients with liver cirrhosis and reduced platelet count (<150/nL). vWF-antigen and vWF-activity were higher in patients with normal results for PFA-100 after measuring with Col-Epi and Col-ADP (A+B). Substitution of recombinant vWF (r-vWF) resulted in improved primary hemostasis as compared to unmodified samples (C). Addition of a 66-µg or 165-µg polyclonal anti-vWF-antibody levels led to prolonged closure times compared to unmodified control samples (D+E). Horizontal lines represent the upper limit of normal for closure times with Col-Epi or Col-ADP [**: <i>P</i><0.01, *: <i>P</i><0.05].</p

Results for univariate analysis of predictors of a pathological PFA-100 test result.

<p>Univariate analysis of the following variables was performed to identify predictors of a pathological PFA-100 test results in the whole study cohort and in thrombocytopenic cirrhotic patients. Multivariate analysis was then performed with variables showing a <i>P</i>-value ≤0.1 in univariate analysis.</p><p>Results for univariate analysis of predictors of a pathological PFA-100 test result.</p