Covalent attachment of maleimide-activated human transferrin to cysteine-modified Ad vectors and their transduction efficiency in hTfR-positive human brain microvascular endothelial cells.

<p>A) Schematic illustration of Ad vector particles containing a solvent-exposed cysteine either on fiber (LIGGG<u>C</u>GGGID) or hexon (HRV5, alanine to cysteine substitution), to which maleimide-activated transferrin is covalently attached (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045977#s4" target="_blank">materials and methods</a>). B–C) Relative transduction efficiency of fiber (AdFiberCys) and hexon-modified (AdHexonCys) vectors with or without covalently attached transferrin in K562 (B) and hCMEC/D3 (C) cells at 24 hrs p.t. by flow cytometry (multiplicities of infection based on particles (pMOIs) 200 and 5000). Relative mean fluorescence and standard deviations are shown (n = 3, 10.000 cells/sample). D) Cellular uptake of the fluid-phase endocytosis marker 70 kDa FITC-Dextran in untransduced K562 and hCMEC/D3 and transduced hCMEC/D3 cells (pMOI 5000) after 1 hr uptake at 37°C determined by flow cytometry. Relative mean fluorescence normalized to untransduced hCMEC/D3 cells, as well as standard deviations are shown (n = 3, 10.000 cells/sample).</p

Delivery of transferrin-coupled hexon-modified Ad vectors across the endothelium barrier.

<p>A) qPCR detection of the transcytosed hTf-coupled vectors after DNAse treatment and viral DNA isolation. Heat and DNAse treated vectors, as well as untreated vectors were used as controls. The percentages were calculated by comparing the detected copy numbers of the untreated vector to the copy numbers of the transcytosed vector. B–C) Gene transfer efficiency of transcytosed hTf-coupled vectors in 293 cells at 16 hrs p.t. as determined by fluorescence microscopy. For quantification, six to eight random areas were imaged and the cells were counted with the help of ImageJ Cell Counter. Transduction units (T.U.) presented were counted from the total sample volume obtained on the basolateral side of the hCMEC/D3 cells (n = 500 cells, mean ± S.E.). Scale bars in all images, 20 µm.</p

Traceless Bioresponsive Shielding of Adenovirus Hexon with HPMA Copolymers Maintains Transduction Capacity In Vitro and In Vivo

<div><p>Capsid surface shielding of adenovirus vectors with synthetic polymers is an emerging technology to reduce unwanted interactions of the vector particles with cellular and non-cellular host components. While it has been shown that attachment of shielding polymers allows prevention of undesired interactions, it has become evident that a shield which is covalently attached to the vector surface can negatively affect gene transfer efficiency. Reasons are not only a limited receptor-binding ability of the shielded vectors but also a disturbance of intracellular trafficking processes, the latter depending on the interaction of the vector surface with the cellular transport machinery. A solution might be the development of bioresponsive shields that are stably maintained outside the host cell but released upon cell entry to allow for efficient gene delivery to the nucleus. Here we provide a systematic comparison of irreversible versus bioresponsive shields based on synthetic <i>N</i>-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In addition, the chemical strategy used for generation of the shield allowed for a traceless bioresponsive shielding, i.e., polymers could be released from the vector particles without leaving residual linker residues. Our data demonstrated that only a bioresponsive shield maintained the high gene transfer efficiency of adenovirus vectors both in vitro and in vivo. As an example for bioresponsive HPMA copolymer release, we analyzed the in vivo gene transfer in the liver. We demonstrated that both the copolymer's charge and the mode of shielding (irreversible versus traceless bioresponsive) profoundly affected liver gene transfer and that traceless bioresponsive shielding with positively charged HPMA copolymers mediated FX independent transduction of hepatocytes. In addition, we demonstrated that shielding with HPMA copolymers can mediate a prolonged blood circulation of vector particles in mice. Our results have significant implications for the future design of polymer-shielded Ad and provide a deeper insight into the interaction of shielded adenovirus vector particles with the host after systemic delivery.</p></div

Delivery of transferrin-coupled fiber-modified Ad vectors across the endothelium barrier.

<p>A, B) After transcytosis experiments in Transwell plates, qPCR was performed from the basolateral media using Ad fiber and E4 primers. The corresponding Ad copy number was determined by the standard curve of linearized pGS66 plasmid. The detected vector copy numbers of fiber- (A) and hexon-modified vectors (B) are shown (2×10⁸, n = 2–3 monolayers; and 1×10⁹ VPs, <i>P</i><0.05, n = 6 monolayers; mean ± S.E.). C) Transcytosis percentages of the vectors after transcellular delivery detected by qPCR across hCMEC/D3 and PBCEC cells (1×10⁹ and 5×10⁹ VPs). The percentages were calculated by comparing the detected copy numbers of the input vector to the copy numbers on the basolateral side.</p

Integrity of hcmec/d3 endothelium in the presence of Ad vectors and their transduction efficiencies in polarized cells.

<p>A) Schematic illustration of the <i>in vitro</i> hCMEC/D3 endothelium model on collagen-coated 0.4 µm-Transwell filters. Cells are grown to confluency for 6–7 days in EBM-2 media, after which the barrier properties of the endothelium are measured by voltohmeter and permeability assays with fluorescent markers. Extracellular matrix (ECM), filter, and apical and basolateral sides of the Transwell chamber are shown. B) Transendothelial electrical resistance (TEER) values of hCMEC/D3 monolayers after 6 days in culture (59.4±1.5 Ω/cm²; mean±S.E., n = 30 monolayers). Boxplot data is shown, containing median (bar), quartile range (box) and minimum and maximum values (whiskers). C) Representative TEER values of hCMEC/D3 monolayers before and after 4 hrs incubation with hexon (AdHexonCys, 5×10⁹ VPs/monolayer) or fiber-modified (AdFiberCys, 1×10⁹ VPs/monolayer) vectors with or without human transferrin (n = 3–4 monolayers/vector). In all experiments, TEER measurements were performed as triplicates with Milli-Cell ERS equipment (mean Ω/cm²±SD, <i>P</i>>0.1). D–E) Transduction efficiencies of fiber or hexon-modified vectors with or without transferrin in unpolarized or transwell-cultured, polarized hCMEC/D3 cells at 24 hrs p.t. detected by fluorimetry (D; n = 3 monolayers, TEER >60 Ω/cm²) or fluorescence microscopy (E; polarized cells). Relative mean fluorescence and standard deviation is calculated from the obtained mean fluorescence values. Scale bars in the images, 50 µm.</p

HPMA copolymers can be released by glutathione.

<p> vector particles were shielded with HPMA copolymers and analyzed by Western blot analysis using a monoclonal anti-hexon antibody. A: Ad was irreversibly shielded with HPMA copolymers # 268 or # 269. Loading buffer contained -mercaptoethanol. B: Ad was shielded with the PySS-activated HPMA copolymers # 279, # 280, # 281 and # 283. Loading buffer contained -mercaptoethanol. C: Ad was shielded with the PySS-activated HPMA copolymer # 281. Before loading, the samples were pre-incubated with different concentrations of glutathione (as indicated) for 30 min at 37. Loading buffer did not contain -mercaptoethanol. Corresponding Western blot analyses with the HPMA copolymers # 279, # 280 and # 283 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082716#pone.0082716.s001" target="_blank">figure S1</a>. Abbreviations: HexCys: unshielded AdHexCys, the “+ Polymer-number” indicates a shielding of AdHexCys with the respective HPMA copolymer, neut.: neutral (uncharged), pos.: positively charged, irrev.: irreversible (mal-)shielding, rev.: bioresponsive (PySS-)shielding.</p

Shielding with bioresponsive HPMA copolymers maintained particle infectivity but reduced FX-mediated transduction.

<p>A: Flow-cytometric analysis of A549 cells 24 h after transduction with 200 pMOI of EGFP-expressing Ad vectors. B: Flow-cytometric analysis of SKOV-3 cells 72 h after infection with 10,000 pMOI of EGFP-expressing Ad vectors in the presence of different concentrations of FX as indicated. Note the grey scale indicating the titration of FX with the white bars indicating absence of FX. The mean fluorescence intensity of unshielded AdHexCys was set to “1” (in B: supplemented with 50 FX) and used to calculate the relative transduction efficiencies of the shielded vector particles. Abbreviations: MFI: mean fluorescence intensity, cont.: untreated control, HexCys: unshielded AdHexCys, the “+ Polymer-number” indicates a shielding of AdHexCys with the respective HPMA copolymer, neut.: neutral (uncharged), pos.: positively charged, irrev.: irreversible (mal-)shielding, rev.: bioresponsive (PySS-) shielding.</p

HPMA copolymer structure and bioresponsive shielding of Ad.

<p>A: Overview over the chemical structures of the HPMA copolymers. HPMA copolymers were activated with either maleimide (mal) groups or pyridyl-dithio (PySS) groups. They were either uncharged (neutral) or were positively charged via a quaternary ammonio groups. B: Reaction of a PySS-activated HPMA copolymer with the thiol group of a cysteine resulting in a traceless bioresponsive shielding of the Ad vector surface. Note the differently colored sulfur atoms indicating the thiol-exchange reaction.</p

Histological cryosections: reduced EGFP expression in the spleen after shielding of Ad with HPMA copolymers.

<p>Female BALB/c were injected with HPMA copolymer-shielded EGFP-expressing Ad vector particles. 72 h later organs were harvested and analyzed by histological cryosections. Magnification: 100-fold, exposure time: liver , spleen , kidney , lung , Abbreviations: HexCys: unshielded AdHexCys, the “+ Polymer-number” indicates a shielding of AdHexCys with the respective HPMA copolymer, neut.: neutral (uncharged), pos.: positively charged, irrev.: irreversible (mal-)shielding, rev.: bioresponsive (PySS-)shielding.</p

Blood circulation kinetics of HPMA copolymer shielded AdHexCys.

<p>To analyze whether shielding with HPMA copolymers would alter the blood circulation kinetics of AdHexCys we injected BALB/c mice with HPMA copolymer-shielded vector particles. 2, 4, 6, 10 and 20 min after vector delivery 20 of blood were taken from the tail vein with a glass capillary by scoring. Total DNA was extracted and Ad genome content was analyzed by QPCR. HPMA copolymers # 268, # 269, # 279 and # 283 not tested. Abbreviations: HexCys: unshielded AdHexCys, the “+ Polymer-number” indicates a shielding of AdHexCys with the respective HPMA copolymer, neut.: neutral (uncharged), pos.: positively charged, rev.: bioresponsive (PySS-) shielding, rel. AUC: relative area under the curve.</p