Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain.

RNA interference (RNAi) for spinocerebellar ataxia type 1 can prevent and reverse behavioral deficits and neuropathological readouts in mouse models, with safety and benefit lasting over many months. The RNAi trigger, expressed from adeno-associated virus vectors (AAV.miS1), also corrected misregulated microRNAs (miRNA) such as miR150. Subsequently, we showed that the delivery method was scalable, and that AAV.miS1 was safe in short-term pilot nonhuman primate (NHP) studies. To advance the technology to patients, investigational new drug (IND)-enabling studies in NHPs were initiated. After AAV.miS1 delivery to deep cerebellar nuclei, we unexpectedly observed cerebellar toxicity. Both small-RNA-seq and studies using AAVs devoid of miRNAs showed that this was not a result of saturation of the endogenous miRNA processing machinery. RNA-seq together with sequencing of the AAV product showed that, despite limited amounts of cross-packaged material, there was substantial inverted terminal repeat (ITR) promoter activity that correlated with neuropathologies. ITR promoter activity was reduced by altering the miS1 expression context. The surprising contrast between our rodent and NHP findings highlight the need for extended safety studies in multiple species when assessing new therapeutics for human application.

Regulated control of gene therapies by drug-induced splicing.

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.

Comparison of changes over time in leukocyte counts in Yucatan minipigs irradiated with simulated solar particle event-like radiation.

During a major solar particle event (SPE), astronauts in space are at risk of exposure to an increased dose of proton radiation. The whole body distribution of the absorbed SPE proton dose is inhomogeneous, and such an inhomogeneous SPE proton dose can be simulated by electron radiation. Using Yucatan minipigs as an animal model, we compared the time courses of leukocyte count changes after exposure to proton simulated SPE (pSPE) radiation or electron simulated SPE (eSPE) radiation. The results demonstrated that the time required after irradiation to reach the lowest leukocyte counts was generally comparable between the pSPE and eSPE radiation exposures. However, the leukocyte count often recovered faster after electron irradiation compared to proton irradiation at the corresponding doses. In addition, the radiation dose required to achieve comparable magnitudes of leukocyte count decrease was higher in the eSPE animals than for the pSPE animals. In conclusion, based on the magnitude of the decrease and the time required to reach the lowest leukocyte counts after irradiation, the pSPE radiation was more effective than the eSPE radiation in reducing the peripheral leukocyte counts. Lymphocytes appeared to be the most sensitive type of leukocytes in response to either type of SPE radiation. It is particularly noteworthy that following exposure to pSPE radiation at the skin doses >5 Gy, the neutrophils do not recover from the radiation damage at times up to 30 days, and the neutrophils have not recovered to their baseline levels even at 90 days post-irradiation. These results suggest a marked difference in the ability of the neutrophils to recover from pSPE radiation compared with the results observed for eSPE radiation.

A combination of metabolic strategies plus cardiopulmonary bypass improves short-term resuscitation from prolonged lethal cardiac arrest.

BACKGROUND: The metabolic or late phase of cardiac arrest is highly lethal. Emergency cardiopulmonary bypass (ECPB) can resuscitate many patients even after prolonged cardiac arrest and provides immediate vascular access for correction of metabolic derangement during the reperfusion process. We developed a rodent model of ECPB resuscitation which showed the superiority of ECPB over conventional CPR, especially when combined with hypothermia. For this study we examined a metabolic strategy against ischemia-reperfusion injury (MS-IR) that included: leukoreduction, low Ca(2+), Mg(2+), buffered pH, red blood cells and a colloid. We tested whether ECPB plus MS-IR and/or hypothermia improves short-term hemodynamic outcomes compared to a standard ECPB reperfusate. METHODS: Using a 2×2 factorial design we tested ECPB with (a) MS-IR versus a standard crystalloid solution; and (b) hypothermia versus normothermia in our rat model. The four reperfusion strategies included: (1) MS-IR plus hypothermia, (2) MS-IR with normothermia, (3) standard plasma-lyte (STD) reperfusate plus hypothermia, or (4) STD plus normothermia. Animals underwent 12 min of untreated asphyxial arrest and were resuscitated with ECPB and one of the four strategies for 30 min. Thereafter, ECPB was discontinued and ventilatory support was provided for 3 hours, while hemodynamic, perfusion and other metrics were serially measured. RESULTS: All rats achieved ROSC with ECPB. Significant differences between the groups emerged after 3 hrs: the best outcomes were in animals with MS-IR plus hypothermia (lactate: 1.1 ± 0.1 mmol/L; MAP: 83 ± 4 mm Hg, seizures: 0/10), while the worst outcomes were with STD and normothermia (lactate: 8.9 ± 1.4 mmol/L, MAP: 36 ± 4 mm Hg, seizures: 7/10, p < 0.001). The outcomes of the other two groups (MS-IR only; hypothermia only) were intermediate. MS-IR and hypothermia improved outcome in an additive fashion. CONCLUSIONS: While most human ECPB is applied with a normothermic crystalloid priming solution, we observed that in rodents the addition of MS-IR plus hypothermia resulted in considerable short-term benefit after prolonged arrest. Future long-term and translational survival studies are warranted to optimize ECPB resuscitation methods.