Impact of Age at Administration, Lysosomal Storage, and Transgene Regulatory Elements on AAV2/8-Mediated Rat Liver Transduction

Liver-directed gene transfer is being investigated for the treatment of systemic or liver-specific diseases. Recombinant vectors based on adeno-associated virus serotype 8 (AAV2/8) efficiently transduce liver cells allowing long term transgene expression after a single administration in animal models and in patients

Spontaneous pneumomediastinum in COVID-19 pneumonia.

Spontaneous pneumomediastinum is a benign entity but can worsen the underlying condition with which it is associated. We evaluated the incidence and the clinical relevance of spontaneous pneumomediastinum in a consecutive series of 102 patients with COVID-19 pneumonia. Six cases of pneumomediastinum were identified by high-resolution chest CT-scan. Three patients required early intubation, and one of them died, while in in the remaining subjects the clinical course was benign. The presence of pneumomediastinum required some changes in the management of mechanical ventilation. In conclusion, spontaneous pneumomediastinum is a possible complication of severe COVID-19 pneumonia that can affect patient management and clinical outcomes

BAG3 protects Bovine Papillomavirus type 1-transformed equine fibroblasts against pro-death signals

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrated the expression of BAG3 protein in equine sarcoids in vivo as well as in an in vitro model of sarcoid-derived equine fibroblasts. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that the most of tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from a healthy horse displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrated the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation in EqSO4b, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome, promotes cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeitic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less relevant in E.Derm cells, taken as normal counterpart, thus suggesting a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival

Lysosomal storage does not impact on AAV2/8-mediated liver transduction.

<p>A) Alcyan blue staining performed on liver sections from wild-type (NR) and MPS VI (AF) rats injected with AAV2/8-TBG-eGFP shows GAG storage (blue staining) in AF but not NR rats. Representative pictures from animals in each group are shown. Magnification: 20×. B) NR and AF rats were injected at P30 with 4×10e13 gc/kg of AAV2/8-TBG-eGFP vectors. Animals were sacrificed at P90 and eGFP expression levels were assessed under a fluorescence microscope on sections from transduced livers. Representative pictures from animals in each group are shown. Magnification: 20×. C) eGFP expression levels were assessed by Western blot analysis of liver lysates from the same animals as in panel B. The intensity of eGFP bands was measured and normalized to that of the corresponding tubulin band. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE. The number of animals analyzed in each group is reported in each bar. D) AAV vector genome copies/molecule of diploid genome (gc/mdg) were measured by Real-time PCR in livers of rats injected with AAV2/8. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE. The number of animals analyzed is reported as before.</p

Noninvasive Repetitive Imaging of Somatostatin Receptor 2 Gene Transfer with Positron Emission Tomography

Noninvasive in vivo imaging of gene expression is desirable to monitor gene transfer in both animal models and humans. Reporter transgenes with low endogenous expression levels are instrumental to this end. The human somatostatin receptor 2 (hSSTR2) has low expression levels in a variety of tissues, including muscle and liver. We tested the possibility of noninvasively and quantitatively monitoring hSSTR2 transgene expression, following adeno-associated viral (AAV) vector-mediated gene delivery to murine muscle and liver by positron emission tomography (PET) using 68gallium-DOTA-Tyr3-Thr8-octreotate (68Ga-DOTATATE) as a highly specific SSTR2 ligand. Repetitive PET imaging showed hSSTR2 signal up to 6 months, which corresponds to the last time point of the analysis, after gene delivery in both transduced tissues. The levels of tracer accumulation measured in muscle and liver after gene delivery were significantly higher than in control tissues and correlated with the doses of AAV vector administered. As repetitive, quantitative, noninvasive imaging of AAV-mediated SSTR2 gene transfer to muscle and liver is feasible and efficient using PET, we propose this system to monitor the expression of therapeutic genes coexpressed with SSTR2

Inclusion of target sequences for miR142-3p in the AAV vector genome does not reduce immune responses to ARSB in MPS VI rats injected with AAV2/8-TBG-hARSB.

<p>MPS VI rats were injected either at P4 or at P30 with AAV2/8-TBG-hARSB or AAV2/8-TBG-hARSB-miR142-Tx4 vectors. Animals were followed up for 6 months after vector delivery when they were sacrificed for analysis of tissue ARSB expression. A) Liver ARSB activity was measured at the time of sacrifice in rats injected at P4 with AAV2/8-TBG-hARSB or AAV2/8-TBG-hARSB-miR142-Tx4. The number of animals analyzed in each group is reported in each bar. NR: control rats AF: uninjected MPS VI rats. * = p<0.05. The p-value between the TBG-hARSB and TBG-hARSB-miR142-Tx4 groups is 0.12. B, C) Serum ARSB activity was measured each month in NR and AF rats (light grey horizontal grids) and in rats injected with either AAV2/8-TBG-hARSB or AAV2/8-TBG-hARSB-miR142-Tx4 vectors at P4 (B) or at P30 (C). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE for each group. D, E) Serum anti-ARSB antibodies were measured by ELISA each month in AF rats injected with either AAV2/8-TBG-hARSB or AAV2/8-TBG-hARSB-miR142-Tx4 vectors at P4 (D) or at P30 (E). As negative control anti-ARSB antibodies were measured in sera from NR and AF rats that did not receive AAV vectors (uninjected controls). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE for each group.</p

Inclusion of the WPRE-m variant is associated with decreased hepatocyte transduction levels in rats.

<p>A) Rats were injected at postnatal day (P)30 with 4×10e13 gc/kg of AAV2/8-TBG-eGFP or AAV2/8-TBG-eGFP-WPRE-m vectors. Animals were sacrificed at P90 and eGFP-positive cells were visualized on liver sections under a fluorescence microscope. Representative pictures from animals in each group are shown. Magnification 20×. B) eGFP expression levels were assessed by Western blot analysis on liver lysates from the same animals as in panel A. The intensity of eGFP bands was measured and normalized on that of the corresponding tubulin band. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE of three independent experiments. The number of animals analyzed in each group is reported inside each bar. *: p<0.05. C) AAV vector genome copies/molecule of diploid genome (gc/mdg) were measured by Real-time PCR in livers of rats injected with AAV. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE of three independent experiments. The number of animals analyzed in each group is reported inside each corresponding bar. The p-value between the TBG-eGFP and TBG-eGFP-WPRE-m groups is 0.44.</p

TBG-driven transgene expression in tissues of rats injected with AAV2/8.

<p>A) Hepatocyte-restricted eGFP expression in the liver of rats injected with AAV2/8-TBG-eGFP. Wild-type rats were injected at P4 (upper pictures), or at P30 (lower pictures) with 4×10e13 gc/kg of AAV2/8-TBG-eGFP vectors. Animals were sacrificed at P15 (those injected at P4) or at P90 (those injected at P30). The identity of eGFP expressing cells in the liver from injected rats was confirmed by confocal microscopy analysis on sections after immuno-fluorescence with anti-albumin (left panels), anti-CD163 (middle panels, white arrows) or anti CD-31 (right panels, white arrows) antibodies. Representative pictures from rats in each group are shown. Magnification: 63×. B, C) Analysis of vector bio-distribution and eGFP expression in tissues from rats injected with AAV2/8-TBG-eGFP. B) AAV vector genome copies/molecule of diploid genome (gc/mdg) were measured in tissues (reported under each bar) from rats injected at P4 (left, for spleen, kidney, muscle, heart and gonads data are pooled from tissues harvested at P15, P30 and P90) or at P30 (right, all tissues harvested at P90) by Real-time PCR. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033286#s2" target="_blank">Results</a> are reported as mean ± SE. The number of animals analyzed in each group is reported in the corresponding bar. C) eGFP expression was assessed by Western blot analysis on the spleen (70 µg of proteins) , kidney (70 µg of proteins) and heart (150 µg of proteins). Representative animals from each group are shown. −: tissues from uninjected rats; +: liver lysate from a rat injected at P4 with AAV2/8-TBG-eGFP and collected at P15 used as eGFP-positive control (10 ug of proteins).</p