Derivation of a Triple Mosaic Adenovirus for Cancer Gene Therapy

A safe and efficacious cancer medicine is necessary due to the increasing population of cancer patients whose particular diseases cannot be cured by the currently available treatment. Adenoviral (Ad) vectors represent a promising therapeutic medicine for human cancer therapy. However, several improvements are needed in order for Ad vectors to be effective cancer therapeutics, which include, but are not limited to, improvement of cellular uptake, enhanced cancer cell killing activity, and the capability of vector visualization and tracking once injected into the patients. To this end, we attempted to develop an Ad as a multifunctional platform incorporating targeting, imaging, and therapeutic motifs. In this study, we explored the utility of this proposed platform by generating an Ad vector containing the poly-lysine (pK), the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK), and the monomeric red fluorescent protein (mRFP1) as targeting, tumor cell killing, and imaging motifs, respectively. Our study herein demonstrates the generation of the triple mosaic Ad vector with pK, HSV-1 TK, and mRFP1 at the carboxyl termini of Ad minor capsid protein IX (pIX). In addition, the functionalities of pK, HSV-1 TK, and mRFP1 proteins on the Ad vector were retained as confirmed by corresponding functional assays, indicating the potential multifunctional application of this new Ad vector for cancer gene therapy. The validation of the triple mosaic Ad vectors also argues for the ability of pIX modification as a base for the development of multifunctional Ad vectors

Facilitated Monocyte-Macrophage Uptake and Tissue Distribution of Superparmagnetic Iron-Oxide Nanoparticles

BACKGROUND: We posit that the same mononuclear phagocytes (MP) that serve as target cells and vehicles for a host of microbial infections can be used to improve diagnostics and drug delivery. We also theorize that physical and biological processes such as particle shape, size, coating and opsonization that affect MP clearance of debris and microbes can be harnessed to facilitate uptake of nanoparticles (NP) and tissue delivery. METHODS: Monocytes and monocyte-derived macrophages (MDM) were used as vehicles of superparamagnetic iron oxide (SPIO) NP and immunoglobulin (IgG) or albumin coated SPIO for studies of uptake and distribution. IgG coated SPIO was synthesized by covalent linkage and uptake into monocytes and MDM investigated related to size, time, temperature, concentration, and coatings. SPIO and IgG SPIO were infused intravenously into naïve mice. T(2) measures using magnetic resonance imaging (MRI) were used to monitor tissue distribution in animals. RESULTS: Oxidation of dextran on the SPIO surface generated reactive aldehyde groups and permitted covalent linkage to amino groups of murine and human IgG and F(ab')(2) fragments and for Alexa Fluor(R) 488 hydroxylamine to form a Schiff base. This labile intermediate was immediately reduced with sodium cyanoborohydride in order to stabilize the NP conjugate. Optical density measurements of the oxidized IgG, F(ab')(2), and/or Alexa Fluor(R) 488 SPIO demonstrated approximately 50% coupling yield. IgG-SPIO was found stable at 4 degrees C for a period of 1 month during which size and polydispersity index varied little from 175 nm and 200 nm, respectively. In vitro, NP accumulated readily within monocyte and MDM cytoplasm after IgG-SPIO exposure; whereas, the uptake of native SPIO in monocytes and MDM was 10-fold less. No changes in cell viability were noted for the SPIO-containing monocytes and MDM. Cell morphology was not changed as observed by transmission electron microscopy. Compared to unconjugated SPIO, intravenous injection of IgG-SPIO afforded enhanced and sustained lymphoid tissue distribution over 24 hours as demonstrated by MRI. CONCLUSIONS: Facilitated uptake of coated SPIO in monocytes and MDM was achieved. Uptake was linked to particle size and was time and concentration dependent. The ability of SPIO to be rapidly taken up and distributed into lymphoid tissues also demonstrates feasibility of macrophage-targeted nanoformulations for diagnostic and drug therapy

Derivation of a Myeloid Cell-Binding Adenovirus for Gene Therapy of Inflammation

The gene therapy field is currently limited by the lack of vehicles that permit efficient gene delivery to specific cell or tissue subsets. Native viral vector tropisms offer a powerful platform for transgene delivery but remain nonspecific, requiring elevated viral doses to achieve efficacy. In order to improve upon these strategies, our group has focused on genetically engineering targeting domains into viral capsid proteins, particularly those based on adenovirus serotype 5 (Ad5). Our primary strategy is based on deletion of the fiber knob domain, to eliminate broad tissue specificity through the human coxsackie-and-adenovirus receptor (hCAR), with seamless incorporation of ligands to re-direct Ad tropism to cell types that express the cognate receptors. Previously, our group and others have demonstrated successful implementation of this strategy in order to specifically target Ad to a number of surface molecules expressed on immortalized cell lines. Here, we utilized phage biopanning to identify a myeloid cell-binding peptide (MBP), with the sequence WTLDRGY, and demonstrated that MBP can be successfully incorporated into a knob-deleted Ad5. The resulting virus, Ad.MBP, results in specific binding to primary myeloid cell types, as well as significantly higher transduction of these target populations ex vivo, compared to unmodified Ad5. These data are the first step in demonstrating Ad targeting to cell types associated with inflammatory disease

MBP fibers are viable and retain myeloid cell-binding specificity.

<p>(<b>A</b>) Diagram comparing wild type Ad5 and the MBP fibers. The MBP fiber is derived from the 566FF platform that our group has previously developed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037812#pone.0037812-Noureddini1" target="_blank">[20]</a>. Briefly, the knob domain is removed and replaced with the trimerization region from the T4 phage fibritin protein fused to the MBP targeting ligand via a flexible linker ([GGGS]₄). (<b>B</b>) Assessment of MBP fiber viability by Western blot. 293T cells were harvested 48 h after transfection with expression plasmids for wild type Ad5, MBP, or inverted MBP (iMBP) fibers. Protein supernatants from 293T cells were incubated at either 95°C (boiled, B) or room temperature (unboiled, U) prior to SDS-PAGE separation. Unboiled samples demonstrate that the majority of fibers are trimerized. (<b>C</b>) Evaluation of fiber binding to CD11b⁺ (myeloid) BMCs from wild type (C57BL/6) and transgenic mice that express hCAR (hCAR Tg). 293T protein supernatants from <b>B</b> were added to BMCs and fiber-bound CD11b⁺ cells were detected by flow cytometry. A representative experiment is shown for <b>C</b>.</p

Phage peptide candidate list.

a<p>Only clones from the third round of <i>in vitro</i> panning were sequenced.</p>b<p>10 minute post-infusion BM harvest.</p>c<p>30 minute post-infusion BM harvest.</p

Identification of a myeloid cell-binding peptide (MBP) by phage display.

<p>(<b>A</b>) Schematic representation of the <i>in vitro</i> and <i>in vivo</i> panning strategies used to identify putative myeloid-binding phage peptides from a cysteine-constrained random heptapeptide M13 phage display library. A complete list of isolated phage peptides is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037812#pone-0037812-t001" target="_blank"><b>Table 1</b></a>. (<b>B</b>) Flow cytometric detection of binding of individual biotinylated-phage clones to bone marrow cells (BMCs) at 4°C. Background binding was assessed with insertless control phage. (<b>C</b>) Flow cytometric detection of binding of WTLDRGY and YGRDLTW phage clones to BMCs at 4°C. Background binding was assessed with insertless control phage. (<b>D</b>) Phage binding to CD11b⁺ BMCs following scanning alanine mutagenesis of the WTLDRGY sequence. Each residue of the WTLDRGY sequence was substituted with an alanine (A) and cloned into the original M13 phage display platform. Phage clones were then amplified and binding was assessed as in <b>B</b> and <b>C</b>. Gray histograms indicate no phage control staining. Representative experiments are shown for <b>B–D</b>.</p

Knob-deleted Ad virus (Ad.MBP and Ad.iMBP) transduction of the CAR-expressing cell line, 293.

<p>Ad5.Luc, Ad.iMBP.Luc, and Ad.MBP.Luc were added to QBI-293A cells in 24-well plates at increasing multiplicities of infection (MOI) for 24 h at 37°C before luciferase activity was assessed at 24 h post-infection. Data are means ± s.d. of a representative experiment with triplicate samples. ##, <i>P</i><0.01; **, <i>P</i><0.01 compared to both Ad5 and Ad.iMBP; ***, <i>P</i><0.001 compared to both Ad5 and Ad.iMBP.</p

Genetic incorporation of MBP fiber into Ad5 (Ad.MBP) retains myeloid cell-binding specificity.

<p>(<b>A</b>) Assessment Ad5-, Ad.iMBP-, and Ad.MBP-binding (2,000 VP per cell) at 4°C to myeloid (CD11b⁺) and non-myeloid (CD11b⁻) BMCs from C57BL/6 and transgenic mice that express hCAR (hCAR Tg) by flow cytometry. (<b>B,C</b>) Evaluation of Ad.MBP-binding to CD11b⁺ BMCs at increasing MOI at 4°C. (<b>B</b>) Percentage of total CD11b⁺ BMCs bound by Ad.MBP. (<b>C</b>) Mean fluorescent intensity (MFI) of Ad.MBP-binding to CD11b⁺ BMCs. (<b>D</b>) Evaluation of Ad5-, Ad.iMBP-, and Ad.MBP-binding (500 VP per cell) at 4°C to murine BM neutrophils was also determined by transmission electron microscopy (TEM). Arrows indicate Ad.MBP virions along the cell surface. Scale bar, 500 nm. (<b>E</b>) Assessment of Ad5- or Ad.MBP-binding (2,000 VP per cell) at 4°C to CD11b⁺ cells from BM, peripheral blood (PB), spleen, and lung by flow cytometry. Binding to CD11b⁻ BMCs is also shown for comparison. Representative experiments are shown for <b>A</b>, <b>D</b>, and <b>E</b>. Data in <b>B</b> and <b>C</b> are means ± s.d. of a representative experiment with triplicate samples.</p