Adaptation of transgene mRNA translation boosts the anticancer efficacy of oncolytic HSV1.

BACKGROUND: Transgenes deliver therapeutic payloads to improve oncolytic virus immunotherapy. Transgenes encoded within oncolytic viruses are designed to be highly transcribed, but protein synthesis is often negatively affected by viral infection, compromising the amount of therapeutic protein expressed. Studying the oncolytic herpes simplex virus-1 (HSV1), we found standard transgene mRNAs to be suboptimally translated in infected cells. METHODS: Using RNA-Seq reads, we determined the transcription start sites and 5'leaders of HSV1 genes and uncovered the US11 5'leader to confer superior activity in translation reporter assays. We then incorporated this 5'leader into GM-CSF expression cassette in oncolytic HSV1 and compared the translationally adapted oncolytic virus with the conventional, leaderless, virus in vitro and in mice. RESULTS: Inclusion of the US11 5'leader in the GM-CSF transgene incorporated into HSV1 boosted translation in vitro and in vivo. Importantly, treatment with US11 5'leader-GM-CSF oncolytic HSV1 showed superior antitumor immune activity and improved survival in a syngeneic mouse model of colorectal cancer as compared with leaderless-GM-CSF HSV1. CONCLUSIONS: Our study demonstrates the therapeutic value of identifying and integrating platform-specific cis-acting sequences that confer increased protein synthesis on transgene expression.

Regulation of autophagy following ex vivo heating in peripheral blood mononuclear cells from young adults.

Under conditions of extreme heat stress, the process of autophagy has previously been shown to protect human cells, but the exact body temperature at which autophagic activation occurs is largely unknown. Further, the interplay between autophagy, the heat shock response (HSR), inflammation, and apoptosis have yet to be examined together under temperature conditions representative of human internal body temperatures at rest (37 °C) or under severe heat stress conditions (41 °C). Thus, the purpose of this study was to examine threshold changes in autophagy, the HSR, inflammation, and apoptosis to increasing levels of ex vivo heat stress. Whole blood was collected from 20 young (23 ± 4 years; 10 men, 10 women) physically active participants. Peripheral blood mononuclear cells (PBMCs) were isolated immediately (baseline) and after 90-min of whole blood heating in 37, 39, and 41 °C water baths, representative of normal resting (non-heat stress) as well as moderate and severe heat stress conditions in humans, respectively. At 37 °C, increased autophagic activity was demonstrated, with no change in the HSR, and inflammation. Subsequently, responses of autophagy, the HSR, and inflammation increased with a moderate heat stress (39 °C), with further increases in only autophagy and the HSR under a severe heat stress of 41 °C. We observed no increase in apoptosis under any temperature condition. Our findings show that in human PBMCs, the autophagy and HSR systems may act cooperatively to suppress apoptotic signaling following heat stress, which may in part be mediated by an acute inflammatory response.

Heat strain in children during unstructured outdoor physical activity in a continental summer climate.

The purpose of this study was to assess the heat strain experienced by children during unstructured physical activity outdoors in a temperate continental summer climate. Eighteen children (7 girls, 12.1 ± 1.7 years) performed up to 4 h of outdoor free-play (duration: 218 ± 33 min; air temperature of 24.5 ± 3.9°C and relative humidity of 66.2 ± 9.2%). Urine specific gravity (USG) was measured pre- and post-free-play, while body core temperature (Tco, ingestible pill) and heart rate (HR) were measured continuously. Physiological strain index (PSI) was calculated from Tco and HR (scale: 0 (none) to 10 (very high)). Activity levels were categorized as rest, light, moderate, and vigorous based on the metabolic equivalent of task, estimated from video analysis. Most children were euhydrated pre (78%, USG ≤ 1.020), but not post-free-play (28%, USG ≤ 1.020). Mean and peak Tco, HR, and PSI responses were 37.8 ± 0.3°C and 38.4 ± 0.3°C, 133 ± 14 bpm and 180 ± 12 bpm, and 4.7 ± 1.1 (low) and 7.4 ± 1.0 (high), respectively. All children reached peak Tco≥38.0°C, with seven ≥38.5°C, and the highest at 38.9°C. The children spent 58 ± 15% of free-play engaged in moderate-to-vigorous intensity physical activity. During free-play, all of the children performed moderate-to-vigorous intensity physical activity, which was associated with pronounced elevations in heat strain.