Corneal Transduction by Intra-Stromal Injection of AAV Vectors In Vivo in the Mouse and Ex Vivo in Human Explants

The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways - but in opposite directions - suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer

The Cell Adhesion Molecule “CAR” and Sialic Acid on Human Erythrocytes Influence Adenovirus In Vivo Biodistribution

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models

Transfection and vector generation.

<p><b>A) Transfection of DKE1 cells and DKSce cells with linear or circular DNA.</b> For each cell line, the level of transfection, based on GFP detection by flow cytometry, was 4–8% for NotI-AscI or I-SceI digested pCAVGFP or pCAVGFP-Sce. Transfection efficiency (±OHTam) increased 7–10 fold when supercoiled 30 kb plasmids were used. <b>B)</b> DKSce cells were transfected with supercoiled pCAVGFP-Sce, I-SceI digested pCAVGFP-Sce or supercoiled pCAVGFP-Sce+OHTam. The transfected cells were collected 5 days later, and the cleared lysate used to infect 10-cm plates of DKSce cells. At this vector generation step, GFP⁺ cells were quantified by flow cytometry and by scanning for fluorescence by microscopy. No GFP⁺ cells were detected when transfecting supercoiled pCAVGFP-Sce without OHTam (non-digested). A non-representative image showing rare GFP expression in I-SceI digested pCAVGFP-Sce (digested), and a representative image showing the GFP expression in supercoiled pCAVGFP-Sce+OHTam (OHTam). Nuclei are stained with Hoechst (blue). Scale bar = 10 µm. *P value = 0.005. <b>C)</b> A 10-cm plate of DKSce cells was incubated with cleared lysate from the above CAVGFP generation step. No GFP⁺ cells were ever detected in the cells transfected with supercoiled pCAVGFP-Sce (non-digested) and reamplified. Approximately 0.2% of the cells were infected by CAVGFP when using the cleared lysate from cells transfected with I-SceI-digested pCAVGFP-Sce (digested). Greater than 10% of the cells in the 10-cm plate were infected with CAVGFP when using the cleared lysate from cells transfected with supercoiled pCAVGFP-Sce+OHTam Nuclei are stained with Hoechst (blue). Scale bar = 10 µm, *P value = 0.029. <b>D)</b> To determine if we could generate vectors more quickly, we repeated the vector generation step using I-SceI digested pCAVGFP-Sce and supercoiled pCAVGFP-Sce+OHTam. The cells were collected at days 2–5 and the cleared lysate was incubated with a fresh monolayer of DKSce cells. The number of CAVGFP infected cells/million transfected cells was quantified. The assays were performed in duplicate and repeated at least three times. <b>E)</b> To determine if we could inhibit or modify the DSB break response, and in turn increase CAV-2 vector generation, we included drugs (caffeine, KU55933, Z-VAD-FMK, and mirin) that play a role in preventing DSB recognition, repair or downstream events. Z-VAD-FMK and mirin were also combined. No significant difference was seen versus controls. The assays were performed in duplicate and repeated at least twice.</p

Transduction efficiency of AAV vectors in the mouse cornea.

<p>EGFP expression (indicated by arrows) in the mouse cornea detected by <i>in vivo</i> epifluorescence microscopy 1-wk post-injection of AAV2/1 (<b>a</b>), AAV2/2 (<b>e</b>), AAV2/5 (<b>i</b>) and AAV2/8 (<b>m</b>) vectors. (<b>b</b>, <b>f</b>, <b>j</b>, <b>n</b>) Higher magnification of <b>a</b>, <b>e</b>, <b>i</b> and <b>m</b>, respectively. (<b>c</b>, <b>g</b>, <b>k</b>, <b>o</b>) EGFP expression in the same corneas detected 4-wk post-injection. (<b>d</b>, <b>h</b>, <b>l</b>, <b>p</b>) Higher magnification of <b>c</b>, <b>g</b>, <b>k</b> and <b>o</b>, respectively. Magnifications <b>a</b>, <b>e</b>, <b>g</b>, <b>i</b>, <b>k</b>, <b>m</b>, <b>o</b>: 20×; <b>b</b>: 43×; <b>c</b>: 25×, <b>d</b>: 53×; <b>f</b>, <b>j</b>: 35×; <b>h</b>: 44×, <b>l</b>: 33×; <b>n</b>: 40×; <b>p</b>: 45×. For reference, the diameter of an adult mouse eye is ∼3.5 mm. The large green spot in centre of photos is the pupil of the mouse eye. The asterisk in panels <b>i</b> to <b>l</b> indicates an opaque lesion on the mouse eye that was present from the beginning of the experiments.</p

Transduction efficiency of the AAV vectors in the human corneal explants.

<p>One-wk post-injection, using <i>in vivo</i> microscopy, EGFP expression can be seen in throughout the cornea following intra-stromal injection of the vectors AAV2/1 (<b>a</b>) and AAV2/8 (<b>i</b>). (<b>b</b>, <b>j</b>) Higher magnification of the boxed regions in <b>a</b> and <b>i</b>, respectively. (<b>e</b>) Three-weeks post transduction, EGFP expression can be detected following AAV2/2 injection. (<b>f</b>) Higher magnification of the boxed area in <b>e</b>. (<b>c</b>, <b>g</b>, <b>k</b>) EGFP expression on histological sections of each cornea 8-wk post-injection of AAV2/1, −/2, −/8, respectively. (<b>d</b>, <b>h</b>, <b>l</b>) Imaris-treated images of <b>c</b>, <b>g</b>, <b>k</b>, respectively, showing EGFP-expressing cells.</p

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways - but in opposite directions - suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer

Cell specificity of AAV2/8 transduction.

<p>(<b>a</b>–<b>d</b>) Anti-CD34 staining (in red) of a non-injected mouse cornea shows abundant CD34+ cells. (<b>e</b>–<b>h</b>) Anti-CD34 labelling (in red) of a mouse cornea 24 h post-injection with 3×10⁹ vg of AAV2/8. Intra-stromal injection results in a decrease in the proportion of CD34+ cells (in red) due to an augmentation in the number of cell nuclei (in blue). The EGFP signal co-localises (arrows and arrowheads) with the CD34-labelled cells. Inset in <b>h</b> shows a higher magnification of the cells indicated by arrowheads minus the Hoechst filter. (<b>i</b>–<b>l</b>) Anti-F4/80 staining (in red) of a non-injected mouse cornea showing a low number of F4/80+ cells. (<b>m</b>–<b>p</b>) Anti-F4/80 labelling (in red) of a mouse cornea 24 h post-injection with 3×10⁹ vg of AAV2/8. The EGFP signal does not co-localise (arrows) with the F4/80-labelled cells.</p

Real-Time PCR analysis of vector genome (a,b,c) and mRNA (d,e,f) levels following AAV2/8 transduction of mouse cornea.

<p>Amplification curves of (<b>a</b>) GAPDH and (<b>b</b>) EGFP in DNA extracted from injected mouse eyes. To aid visualisation, only one of the duplicate curves is shown. (<b>c</b>) Graphical representation of the concentration of EGFP normalised to that of GAPDH in each sample (the colour of the curves in <b>a and b</b> matches those of the corresponding bars in <b>c</b>). As a control, EGFP DNA could not be detected following injection and re-injection of the mouse cornea with PBS (PBS PBS). In contrast, EGFP DNA levels were detected and relatively stable both 8- and 14-d post-AAV2/8 transduction (blue bars). Following a second injection of PBS 7-d post-transduction, a 1.9- and 4-fold decrease in EGFP DNA was detected on days 8 and 14, respectively (red bars). Amplification curves of (<b>d</b>) GAPDH and (<b>e</b>) EGFP in RNA extracted from injected mouse eyes. (<b>f</b>) Graphical representation of the concentration of EGFP normalised to that of GAPDH in each sample (the colour of the curves in <b>d and e</b> matches those of the corresponding bars in <b>f</b>). As a control, EGFP RNA levels could not be detected following injection and re-injection of the mouse cornea with PBS. Similarly, EGFP RNA levels could not be detected 8- and 14-d post AAV2/8 transduction. In contrast, a second injection of PBS 7-d post-transduction resulted in a 65-fold increase in EGFP RNA levels on day 8 (red bar). RNA levels were no longer detectable by day 14.</p

EGFP-expressing cell population following PBS injection in the transduced mouse cornea.

<p>(<b>a</b>–<b>d</b>) Anti-CD34 staining (in red) of a mouse cornea 24 h post-PBS injection (same eye shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035318#pone-0035318-g004" target="_blank">Fig. 4C</a>). The CD34 signal co-localises with the EGFP signal (arrows and arrowheads). (<b>e</b>–<b>f</b>) Anti-F4/80 staining (in red) of a mouse cornea 24 h post-PBS injection. The F4/80 signal does not co-localise with the EGFP signal (arrows).</p