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

Corrective GUSB transfer to the canine mucopolysaccharidosis VII cornea using a helper-dependent canine adenovirus vector.

International audienceCorneal transparency is maintained, in part, by specialized fibroblasts called keratocytes, which reside in the fibrous lamellae of the stroma. Corneal clouding, a condition that impairs visual acuity, is associated with numerous diseases, including mucopolysaccharidosis (MPS) type VII. MPS VII is due to deficiency in β-glucuronidase (β-glu) enzymatic activity, which leads to accumulation of glycosaminoglycans (GAGs), and secondary accumulation of gangliosides. Here, we tested the efficacy of canine adenovirus type 2 (CAV-2) vectors to transduce keratocyte in vivo in mice and nonhuman primates, and ex vivo in dog and human corneal explants. Following efficacy studies, we asked if we could treat corneal clouding by the injection a helper-dependent (HD) CAV-2 vector (HD-RIGIE) harboring the human cDNA coding for β-glu (GUSB) in the canine MPS VII cornea. β-Glu activity, GAG content, and lysosome morphology and physiopathology were analyzed. We found that HD-RIGIE injections efficiently transduced coxsackievirus adenovirus receptor-expressing keratocytes in the four species and, compared to mock-injected controls, improved the pathology in the canine MPS VII cornea. The key criterion to corrective therapy was the steady controlled release of β-glu and its diffusion throughout the collagen-dense stroma. These data support the continued evaluation of HD CAV-2 vectors to treat diseases affecting corneal keratocytes

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

IL-21 promotes the development of a CD73-positive Vγ9Vδ2 T cell regulatory population

International audienceVγ9Vδ2 T cells contribute to the immune response against many tumor types through their direct cytotoxic activity and capacity to regulate the biological functions of other immune cells, such as dendritic cells and IFN-γ-producing CD8+ T cells. However, their presence in the tumor microenvironment has also been associated with poor prognosis in breast, colon and pancreatic cancers. Additionally, recent studies demonstrated that cytokines can confer some plasticity to Vγ9Vδ2 T cells and promote their differentiation into cells with regulatory functions. Here, we demonstrated that activation of Vγ9Vδ2 T cells isolated from healthy donors and cultured in the presence of IL-21 favors the emergence of a subpopulation of Vγ9Vδ2 T cells that express the ectonucleotidase CD73 and inhibits T cell proliferation in a CD73/adenosine-dependent manner. This subpopulation produces IL-10 and IL-8 and displays lower effector functions and cytotoxic activity than CD73-negative Vγ9Vδ2 T cells. We also showed, in a syngeneic mouse tumor model, the existence of a tumor-infiltrating γδ T cell subpopulation that produces IL-10 and strongly expresses CD73. Moreover, maturation, IL-12 production and induction of antigen-specific T cell proliferation are impaired in DC co-cultured with IL-21-amplified Vγ9Vδ2 T cells. Altogether, these data indicate that IL-21 promotes Vγ9Vδ2 T cell regulatory functions by favoring the development of an immunosuppressive CD73+ subpopulation. Thus, when present in the tumor microenvironment, IL-21 might negatively impact γδ T cell anti-tumor functions

Corrective GUSB transfer to the canine mucopolysaccharidosis VII brain

International audienceSevere deficiency in lysosomal β-glucuronidase (β-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in β-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the \textgreater200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, β-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases

CAV-2-Mediated GFP and LRRK2G2019S Expression in the Macaca fascicularis Brain

Parkinson’s disease is characterized by motor and nonmotor symptoms that gradually appear as a consequence of the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Currently, no treatment can slow Parkinson’s disease progression. Inasmuch, there is a need to develop animal models that can be used to understand the pathophysiological mechanisms underlying dopaminergic neuron death. The initial goal of this study was to determine if canine adenovirus type 2 (CAV-2) vectors are effective gene transfer tools in the monkey brain. A second objective was to explore the possibility of developing a large nonhuman primate that expresses one of the most common genetic mutations causing Parkinson’s disease. Our studies demonstrate the neuronal tropism, retrograde transport, biodistribution, and efficacy of CAV-2 vectors expressing GFP and leucine-rich repeat kinase 2 (LRRK2G2019S) in the Macaca fascicularis brain. Our data also suggest that following optimization CAV-2-mediated LRRK2G2019S expression could help us model the neurodegenerative processes of this genetic subtype of Parkinson’s disease in monkeys

Downregulation of endocytotic and Wnt genes in HD-HAd cells.

<p>(<b>a</b>) <i>CLTC</i> and <i>MYO6</i> modulations were evaluated by qPCR at 2 h, 5 and 10 days posttransduction. Data are presented as fold change expression of each transcript normalized with respect to the endogenous control and calculated as compared to the mock sample considered as 1. Red dashed lines display the threshold set at ±1.5, as for microarray data. Data are reported as mean with SD. All samples were tested in triplicate; one-way ANOVA (vectors versus mock), ** p<0.01, , *** p<0.001. (<b>b</b>) Wnt/TGF-β pathway in HD-HAd cells 2 h posttransduction. In red are highlighted the upregulated genes, in green the downregulated ones, in white genes not modulated in the chip; IPA p-value 4.01×10⁻². A detailed legend is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069808#pone-0069808-g004" target="_blank">figure 4c</a>. (<b>c</b>) <i>FXN</i> modulation quantified by qPCR at 2 h, 5 and 10 days days posttransduction. Data are presented as fold change expression of each transcript normalized with respect to the endogenous control as in (a).</p

Comparative analysis of cell cycle and DNA damage gene expression profiles induced by HD-HAd, HD-CAV-2 and LV.

<p>(<b>a–c</b>) Heat maps of genes identified by combined IPA and GO analysis with a threshold set at p≤0.01. The relative gene modulation fold change values are indicated; in red, upregulated genes, in green, downregulated, and in grey, genes with unmodified expression with respect to mock. The most prominent trait of this analysis is the divergent modulation of the subgroup GO: 00006974 (“Regulation of DNA damage stimulus”) and GO: 0010564 (“Regulation of cell cycle process”) by HD-CAV-2 and LV at 5 days posttransduction.</p

hmNPCs differentiation and transduction.

<p>(<b>a</b>) qPCR quantification of the markers GFAP, TUJ-1 and TH in differentiated hmNPCs presented as fold changes relative to undifferentiated cells. Values are averages from two independent experiments and SD is shown. p-values are determined by a two-tailed paired Student's t test from the ΔCq values of differentiated with respect to the undifferentiated condition; ** p<0.01. (<b>b</b>) qPCR quantification of internalized vector copies performed on differentiated hmNPCs transduced with HD-HAd, HD-CAV-2 and LV vectors at an MOI of 1000 vector genomes/cell. Results are presented as average of two independent experiments; SD is shown. Data were compared by one-way ANOVA followed by Tukey's post- hoc test between vectors for each time point; * p<0.05. (<b>c–d</b>) Differentiated hmNPCs were transduced with HD-HAd, HD-CAV-2 and LV at an MOI of 1000 vector genomes/cell and were analysed for GFP expression by FACS at 5 days (c), and by qPCR at 5 and 10 days (d). FACS results are representative of three independent experiments. qPCR data are the average of triplicate samples and are presented as fold change values of 10 days values versus 5 days. SD is shown. (<b>e</b>) Schematic representation of chip analysis workflow. (<b>f</b>) Scatter plots showing on the y axis the Robust Multichip Average (RMA) expression values for mock cells, and in x RMA values for of HD-HAd, HD-CAV-2 and LV transduced samples, at the 2 h and 5 days posttransduction time points. To obtain RMA, background corrected raw intensity values were Log2 transformed and quantile normalized. All absent and control probes were excluded from this analysis. Values represent the average of the three independent replica experiments. Trend lines and R² value are indicated. (<b>g</b>) Number of genes upregulated and downregulated with a fold change ≤−1.5 or ≥+1.5 in response to HD-HAd, HD-CAV-2 and LV at 2 h and 5 days. Red: upregulated genes; green: downregulated genes. (<b>h</b>) Heat maps of genes commonly modulated by HD-HAd, HD-CAV-2 and LV and at 2 h and 5 days posttransduction. The relative fold change values are indicated; in red, upregulated genes, in green, downregulated, and in grey, genes with unmodified expression with respect to mock. (<b>i</b>) Correlation between relative fold changes measured by microarray and qPCR. Each point represents the fold change of a single gene relative to the mock at a given time point; x and y axes are microarray and qPCR fold changes, respectively. Number of xy pairs = 28. Pearson's <i>r</i> correlation = 0.75; p<0.0001 (2-tailed). All samples were tested in triplicate and data are reported as mean value. Tested genes were the following: <i>FANCD2</i>, <i>BIRC5</i>, <i>MAD2L1</i>, <i>RAD51</i>, <i>NBN</i>, <i>HIP1B</i>, <i>MYO6</i>, <i>CLTC</i>, <i>CD44</i>, <i>TLR3</i>,<i>TLR4</i>, <i>HAS3</i>, <i>FXN</i>, <i>p53</i>, <i>CDKN1A.</i></p

Comparative analysis of immune response gene expression profiles induced by HD-HAd, HD-CAV-2 and LV.

<p>(<b>a–d</b>) Heat maps of genes identified by combined IPA and GO analysis with a threshold set at p≤0.01, clustering in immune response categories. The relative gene modulation fold change values are indicated; in red, upregulated genes, in green, downregulated, and in grey, genes with unmodified expression with respect to mock. Of note is the LV dependent activation of the IFN subgroup (GO: 0060337) and the LV and HD-CAV-2 induced activation of the TLRs and HA related signaling pathway components at 5 days posttransduction.</p