Lipid nanoparticle-encapsulated, chemically modified anti-adenoviral siRNAs inhibit hepatic adenovirus infection in immunosuppressed Syrian hamsters

RNA interference has demonstrated its potential as an antiviral therapy for treatment of human adenovirus (hAd) infections. The only existing viral vector-based system for delivery of anti-adenoviral artificial microRNAs available for in vivo use, however, has proven to be inefficient in therapeutic applications. In this study, we investigated the potential of stabilized small interfering RNA (siRNA) encapsulated in lipid nanoparticles (LNPs) for treatment of hepatic hAd serotype 5 (hAd5) infection in an hAd infection model using immunosuppressed Syrian hamsters. The siRNA sipTPmod directed against the adenoviral pre-terminal protein (pTP) and containing 2′-O-methyl modifications as well as phosphorothioate linkages effectively inhibited hAd5 infection in vitro. In light of this success, sipTPmod was encapsulated in LNPs containing the cationic lipid XL-10, which enables hepatocyte-specific siRNA transfer, and injected intravenously into hAd5-infected immunosuppressed Syrian hamsters. This resulted in a significant reduction of liver hAd5 titers, a trend toward reduced liver injury and inflammation, and reduction of viral titers in the blood and spleen compared with hAd5-infected animals that received a non-silencing siRNA. These effects were demonstrated in animals infected with low and moderate doses of hAd5. These data demonstrate that hepatic hAd5 infection can be successfully treated with anti-adenoviral sipTPmod encapsulated in LNPs

Silencing of Mcl-1 overcomes resistance of melanoma cells against TRAIL-armed oncolytic adenovirus by enhancement of apoptosis

Arming of oncolytic viruses with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown as a viable approach to increase the antitumor efficacy in melanoma. However, melanoma cells may be partially or completely resistant to TRAIL or develop TRAIL resistance, thus counteracting the antitumor efficiency of TRAIL-armed oncolytic viruses. Recently, we found that TRAIL resistance in melanoma cells can be overcome by inhibition of antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1). Here, we investigated whether the cytotoxicity of AdV-TRAIL, an oncolytic adenovirus, which expresses TRAIL after induction by doxycycline (Dox), can be improved in melanoma cells by silencing of Mcl-1. Two melanoma cell lines, the TRAIL-resistant MeWo and the TRAIL-sensitive Mel-HO were investigated. Treatment of both cell lines with AdV-TRAIL resulted in a decrease of cell viability, which was caused by an increase of apoptosis and necrosis. The proapoptotic effects were dependent on induction of TRAIL by Dox and were more pronounced in Mel-HO than in MeWo cells. SiRNA-mediated silencing of Mcl-1 resulted in a further significant decrease of cell viability and a further increase of apoptosis and necrosis in AdV-TRAIL-infected MeWo and Mel-HO cells. However, while in absolute terms, the effects were more pronounced in Mel-HO cells, in relative terms, they were stronger in MeWo cells. These results show that silencing of Mcl-1 represents a suitable approach to increase the cytotoxicity of a TRAIL-armed oncolytic adenovirus in melanoma cells.TU Berlin, Open-Access-Mittel – 202

Application Route and Immune Status of the Host Determine Safety and Oncolytic Activity of Oncolytic Coxsackievirus B3 Variant PD-H

The coxsackievirus B3 strain PD-0 has been proposed as a new oncolytic virus for the treatment of colorectal carcinoma. Here, we generated a cDNA clone of PD-0 and analyzed the virus PD-H, newly generated from this cDNA, in xenografted and syngenic models of colorectal cancer. Replication and cytotoxic assays revealed that PD-H replicated and lysed colorectal carcinoma cell lines in vitro as well as PD-0. Intratumoral injection of PD-H into subcutaneous DLD-1 tumors in nude mice resulted in strong inhibition of tumor growth and significantly prolonged the survival of the animals, but virus-induced systemic infection was observed in one of the six animals. In a syngenic mouse model of subcutaneously growing Colon-26 tumors, intratumoral administration of PD-H led to a significant reduction of tumor growth, the prolongation of animal survival, the prevention of tumor-induced cachexia, and the elevation of CD3+ and dendritic cells in the tumor microenvironment. No virus-induced side effects were observed. After intraperitoneal application, PD-H induced weak pancreatitis and myocarditis in immunocompetent mice. By equipping the virus with target sites of miR-375, which is specifically expressed in the pancreas, organ infections were prevented. Moreover, employment of this virus in a syngenic mouse model of CT-26 peritoneal carcinomatosis resulted in a significant reduction in tumor growth and an increase in animal survival. The results demonstrate that the immune status of the host, the route of virus application, and the engineering of the virus with target sites of suitable microRNAs are crucial for the use of PD-H as an oncolytic virus

Application Route and Immune Status of the Host Determine Safety and Oncolytic Activity of Oncolytic Coxsackievirus B3 Variant PD-H

The coxsackievirus B3 strain PD-0 has been proposed as a new oncolytic virus for the treatment of colorectal carcinoma. Here, we generated a cDNA clone of PD-0 and analyzed the virus PD-H, newly generated from this cDNA, in xenografted and syngenic models of colorectal cancer. Replication and cytotoxic assays revealed that PD-H replicated and lysed colorectal carcinoma cell lines in vitro as well as PD-0. Intratumoral injection of PD-H into subcutaneous DLD-1 tumors in nude mice resulted in strong inhibition of tumor growth and significantly prolonged the survival of the animals, but virus-induced systemic infection was observed in one of the six animals. In a syngenic mouse model of subcutaneously growing Colon-26 tumors, intratumoral administration of PD-H led to a significant reduction of tumor growth, the prolongation of animal survival, the prevention of tumor-induced cachexia, and the elevation of CD3+ and dendritic cells in the tumor microenvironment. No virus-induced side effects were observed. After intraperitoneal application, PD-H induced weak pancreatitis and myocarditis in immunocompetent mice. By equipping the virus with target sites of miR-375, which is specifically expressed in the pancreas, organ infections were prevented. Moreover, employment of this virus in a syngenic mouse model of CT-26 peritoneal carcinomatosis resulted in a significant reduction in tumor growth and an increase in animal survival. The results demonstrate that the immune status of the host, the route of virus application, and the engineering of the virus with target sites of suitable microRNAs are crucial for the use of PD-H as an oncolytic virus

Functional and Morphological Cardiac Magnetic Resonance Imaging of Mice Using a Cryogenic Quadrature Radiofrequency Coil

<div><p>Cardiac morphology and function assessment by magnetic resonance imaging is of increasing interest for a variety of mouse models in pre-clinical cardiac research, such as myocardial infarction models or myocardial injury/remodeling in genetically or pharmacologically induced hypertension. Signal-to-noise ratio (SNR) constraints, however, limit image quality and blood myocardium delineation, which crucially depend on high spatial resolution. Significant gains in SNR with a cryogenically cooled RF probe have been shown for mouse brain MRI, yet the potential of applying cryogenic RF coils for cardiac MR (CMR) in mice is, as of yet, untapped. This study examines the feasibility and potential benefits of CMR in mice employing a 400 MHz cryogenic RF surface coil, compared with a conventional mouse heart coil array operating at room temperature. The cryogenic RF coil affords SNR gains of 3.0 to 5.0 versus the conventional approach and hence enables an enhanced spatial resolution. This markedly improved image quality – by better deliniation of myocardial borders and enhanced depiction of papillary muscles and trabeculae – and facilitated a more accurate cardiac chamber quantification, due to reduced intraobserver variability. In summary the use of a cryogenically cooled RF probe represents a valuable means of enhancing the capabilities of CMR of mice.</p> </div

End-diastole (a) and end-systole (b) short axis views acquired using the CryoProbe together with a ultra-high spatial resolution of (43×138×300) µm³.

<p>Endo- and epicardial border delineation as well as the depiction of anatomic details of the papillary muscles and trabeculae are improved versus the high resolution CryoProbe images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042383#pone-0042383-g003" target="_blank">Figure 3</a> e,f) due to the enhanced spatial resolution used in the ultra-high spatial resolution protocol.</p

<i>a,b:</i> Bar plot of mean LV myocardium signal-to-noise ratio (SNR) (b) measured in images acquired with the CryoProbe (CP) or room temperature coil (RT) for all mice in the different segments of a six-segment model (a).

<p>The segments were numbered clockwise, starting at the inferoseptal segment. The region closest to the coil (segment 3) showed the highest SNR, and SNR decreased with distance from the coil. <i>c,d</i>: Bar plot of mean LV myocardium SNR (d) for all mice in the different slices (geometry shown in c). Slices were numbered from the basal slice to the apex. For the CryoProbe the apical slice showed an unexpected increase in SNR, in contrast to the decreasing trend from base to apex in the remaining 6 slices.</p

Comparison of end-diastole (left column) and end-systole (right column) short axis views acquired using a spatial resolution of 156×234×800 µm³ (a,b) and 69×115×800 µm³ (c,d,e,f).

<p>The depiction of anatomic details for left ventricular papillary muscles and right ventricular trabeculae is enhanced in the CryoProbe (CP) images (e,f) compared to the room temperature (RT) coil images (a,b,c,d) for both diastole and systole. Coronary arteries are more pronounced in the CryoProbe images. The lateral right endocardial boundary is better delineated in the CryoProbe images. The signal-to-noise ratio of the lower resolved images acquired with the RT-coil (47±9, a,b) is comparable to that of the higher resolved images acqured with the CryoProbe (54±7, e,f).</p