CERE-120 Prevents Irradiation-Induced Hypofunction and Restores Immune Homeostasis in Porcine Salivary Glands

Salivary gland hypofunction causes significant morbidity and loss of quality of life for head and neck cancer patients treated with radiotherapy. Preventing hypofunction is an unmet therapeutic need. We used an adeno-associated virus serotype 2 (AAV2) vector expressing the human neurotrophic factor neurturin (CERE-120) to treat murine submandibular glands either pre- or post-irradiation (IR). Treatment with CERE-120 pre-IR, not post-IR, prevented hypofunction. RNA sequencing (RNA-seq) analysis showed reduced gene expression associated with fibrosis and the innate and humoral immune responses. We then used a minipig model with CERE-120 treatment pre-IR and also compared outcomes of the contralateral non-IR gland. Analysis of gene expression, morphology, and immunostaining showed reduced IR-related immune responses and improved secretory mechanisms. CERE-120 prevented IR-induced hypofunction and restored immune homeostasis, and there was a coordinated contralateral gland response to either damage or treatment. CERE-120 gene therapy is a potential treatment for head and neck cancer patients to influence communication among neuronal, immune, and epithelial cells to prevent IR-induced salivary hypofunction and restore immune homeostasis

Adenovirus Gene Transfer to Amelogenesis Imperfecta Ameloblast-Like Cells

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including “pK7” and/or “RGD” motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber “knob” domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both αvβ3/αvβ5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI

Therapeutic Effects of Environmental Enrichment on Cognitive Function and Tissue Integrity Following Severe Traumatic Brain Injury in Rats

Postinjury environmental enrichment (EE) has been shown to alter functional and anatomical outcomes in a number of injury paradigms, including traumatic brain injury (TBI). The question of whether EE alters functional outcome following TBI in a model which produces overt histopathological consequences has not been addressed. We investigated this question using the severe, parasagittal fluid percussion injury (FPI) model. Rats (n = 7 per group, enriched and standard for behavior; n = 15 per group for histology) underwent severe (2.2–2.6 atm) FPI, with sham-operated rats (n = 7 per group, enriched and standard for behavior; n = 6 enriched, n = 3 standard for histology) serving as controls. Animals were allowed to recover for 11 days either in standard single housing or together (injured and sham) in an enriched environment consisting of a 92 × 61 × 77-cm ferret cage filled with various stimulatory objects. Consistent with earlier reports, injured animals recovering in the enriched environment showed significantly (P < 0.05) shorter latencies to find the platform in a Morris Water Maze task versus injured/standard animals on day 12 post-TBI. However, both injured groups showed significant deficits versus sham groups (P < 0.05). There were no differences between the sham/enriched and sham/standard groups. No significant group differences in swim speed were observed. At 14 days post-TBI, enriched animals had approximately twofold smaller lesion areas in regions of the cerebral cortex posterior to the injury epicenter (−4.5, −5.8, −6.8 mm relative to bregma; P < 0.05) compared to injured/standard animals. In addition, overall lesion volume for the entire injured cortical hemisphere was significantly smaller in animals recovering in the enriched environment. These results indicate that noninvasive environmental stimulation is beneficial in attenuating cognitive deficits and preserving tissue integrity in a TBI model which causes cerebral contusion and cell death

Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis

Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis

Schematic representation of the Ad5 fiber proteins carrying intact and modified C-terminal knob domains.

<p>Fiber modifications are indicated in the corresponding vector names: Ad5 (G/L) has unmodified fiber knob and possesses the native CAR tropism; Ad5-RGD contains a peptide ligand with an “RGD motif” in the HI loop (red loop) of the fiber knob; Ad5-pK7 contains a stretch of seven lysine residues (green oval) fused to the C-terminus of the Ad5 knob via a (GS)₅ linker (green hook); Ad5-pK7/RGD incorporates both modifications in the corresponding locales of the same fiber molecule; Ad5/3 contains a chimera fiber with Ad5 fiber “knob” domain (gray) replaced with the Ad serotype 3 (Ad3) knob (blue), which retargets the vector to Ad3 receptor(s).</p

Ad5 gene transfer to AI-WAm cells is limited by deficiency in expression and/or cell surface localization of the Ad5 receptor CAR.

<p><b>A.</b> Gene transfer efficiency of a human Ad5 vector expressing Luc reporter (Ad5 (L)) to an AI patient-derived ameloblast-like cells (AI-WAm) at different multiplicities of infection (MOI) (MOI = 10, 50 and 250 TCID₅₀/cell) in comparison to CAR-positive A549 and CAR-negative RD cells by conventional Luc assay at 20 hours post infection. Results are presented in Relative Luc Units (RLU) per cell with mean values shown above each bar plus/minus standard deviation. All differences were statistically significant (<i>P</i><0.05) <b>B.</b> Expression levels of hCAR mRNA in AI-WAm and A549 cells relative to that in RD cells as determined by qRT-PCR and presented as “fold difference”. All differences were statistically significant (P<0.05). <i>P_{(A549/AI-WAm)} = 0.027</i>. <b>C.</b> Quantitative analysis of hCAR and hCD46 mRNA expression levels in RD, AI-WAm and A549 cells as determined by qRT-PCR and normalized to the housekeeping gene <i>GAPDH</i>. The data are presented as ΔΔCt values. For AI-WAm <i>P_(CAR/CD46)</i> = 0.44; for A549 <i>P_(CAR/CD46)</i> = 0.127; <i>for RD P_(CAR/CD46)</i> = 0.007; for CAR <i>P_{(AI-WAm/A549)} = 0.049</i>; <i>P_(RD/AI-WAm) = 0.033</i>; for CD46 <i>P_(RD/AI-WAm) = 0.0011</i>; <i>P_{(AI-WAm/A549)} = 0.0008</i>. <i>P</i>-values for all other differences were <0.05. <b>D.</b> Flow cytometry analysis of Ad5 receptor (CAR) expression in AI-WAm cell population in comparison with CAR-positive (A549) and CAR-negative (RD) control cells. Cells were incubated with primary anti-CAR (RmcB) monoclonal antibody (Ab) followed by labeling with Alexa 488-conjugated secondary antibodies. No primary Ab was used in negative control samples. The extent of shift in the fluorescent peak positions (color lines) relative to control peak(s) of unlabeled cells (black dotted lines) reflects the extent of cell labeling, corresponding to the receptor expression on each cell type and is expressed as Mean Fluorescence Intensity (MFI). Numbers above each peak correspond to percentage (%) of gated (M2) cells calculated using subjective gating. Fluorescence intensity (X-axis) is plotted as histograms on log scale (X-axis) using Flowjo 7.6.4 software (Tree Star Inc., Ashland OR). Y-axis depicts total events (cells) and expressed either as counts or % of maximal. <i>P_{(AI-WAm/A549)} = 0.0038</i>; <i>P_(AI-WAm/RD) = 0.39</i>; <i>P_(A549/RD) = 0.0018</i>; <i>P_{(A549/HEK293T)} = 0.018</i>; CAR <i>P_{(AI-WAm/HEK293)} = 0.0001</i>; <i>P_(HEK293/RD) = 0.001</i>; <b>E.</b> Comparison of hCAR expression in AI-WAm and control cells by IHC staining. CAR-specific (RmcB) primary antibody (same as used for FACS analysis, D) was used to stain AI-WAm cells. A549 and HEK293-T cells were used as positive and RD cells as negative controls for CAR expression. All cells were counter-stained for 5 min. with 300 nM DAPI to visualize nuclear DNA (blue). No primary antibody was used with control samples. Negative control (RD) cells show efficient nuclear staining but no discernible CAR staining (green), while A549 cells demonstrate a strong CAR-specific signal and AI-WAm cells display a moderate level of hCAR signal with diffuse pattern of cytosolic localization (white arrows) similar to that in A549 cells. In sharp contrast, CAR-overexpressing HEK293-T cells show a distinct localization of CAR protein in the cell membrane tight junctions (red arrows) with lesser cytosolic staining. Scale bars correspond to 100 µm.</p

The infectivity enhancement effect of fiber-modified Ad5 vectors is mediated by α_vβ3/α_vβ5 integrins and/or HSPG molecules on AI-ameloblasts.

<p><b>A.</b> Differential blocking of gene transfer to AI-WAm cells by integrins. Ad5 RGD shows the highest sensitivity to integrin blocking, while transduction with Ad5-pK7/RGD (G/L) is only partially inhibited. Ad5-pK7 (G/L) gene transfer shows no statistically significant inhibition by integrins. <b>B.</b> Blocking of AI-WAm gene transfer by modified vectors with heparin. Heparin shows a profound dose-dependent blocking effect on transduction with pK7-modified Ads, as opposed to RGD-modified vector. Gray bars (with % values on the top) show percentage of the residual gene transfer level (RLU) resulting from blocking relative to that of unblocked controls (100%) shown by black bar for each fiber-modified vector. All bars represent mean values with standard deviations. All differences were statistically significant except where indicated by asterisk and <i>P</i> values (<i>P</i>>0.05) on the data bars.</p

Expression analyses of HSPG and integrin molecules as alternate receptors for fiber-modified Ads on AI-WAm cells.

<p>Expression of α_vβ3 and α_vβ5 integrins (<b>A</b> and <b>B</b>) and heparin sulfate proteoglycans (<b>C–E</b>) in AI-WAm cell population and control cells was analyzed by flow cytometry (<b>A</b>, <b>C</b>, <b>E</b>) and IHC staining (<b>B</b> and <b>D</b>). Cells were incubated with primary anti-α_vβ3 or anti-α_vβ5 monoclonal antibodies for detection of corresponding integrin molecules or 10E4 antibody for detection of HSPG side chains (GAG) or anti-human syndecan 4 monoclonal antibody, followed by Alexa 488-conjugated secondary antibody. <b>A</b> and <b>C</b>, top charts: AI-WAm cells; middle charts: RD cells; bottom charts: A549 cells. For AI-WAm cells: <i>P</i>_{(αvβ3/αvβ5)} = <i>0.75</i>; <i>P</i>_(Synd4/HSPG) = <i>0.29</i>; For RD cells: <i>P</i>_(Synd4/HSPG) = <i>0.67</i>; <i>for</i> α_vβ3: <i>P_(RD/A549) = 0.38</i>; <i>for</i> α_vβ5: <i>P_{(AI-WAm/A549)} = 0.23</i>; <i>for syndecan 4: P</i>_{(<i>RD</i>/A549)} = <i>0.2</i>; <i>for</i> all other differences <i>P</i><0.05; <b>E.</b> HSPG Ab (10E4) specificity control sample: A549 cells were treated with heparitinase (10 U/ml) for 1 hr at 37°C to remove GAG side chains. Green arrow shows shift of the fluorescence intensity peak resulting from reduction in cell labeling with 10E4 antibody (MFI decrease). Other details are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024281#pone-0024281-g002" target="_blank"><b>Fig. 2D</b></a>. <b>B</b> and <b>D</b>, scale bars correspond to: 100 µm in top image panels (integrins/AI-WAm, 10× objective), 10 µm (insert, 60× objective) and 50 µm (40× objective) in all other panels. Insert shows syndecan 4 staining image (60×) of A549 cells, clearly demonstrating a polarized intracellular localization of the protein.</p