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Luciferase expression
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Luciferase expression was detectable as early as day 3 after injection with the AAVHSC15 Rosa26-Luc editing vector, and was stable to day 112 postinjection, the last time point assayed. Expression gradually increased after injection and plateaued within ∼4–6 wk. Luciferase expression was observed systemically, consistent with the expected ubiquitous expression of Rosa26 (69). In vivo imaging indicated strong widespread systemic luciferase expression (Fig. 5B). Organ-specific expression was assessed in isolated organs at the end of the experiment. Quantitation of flux in isolated organs revealed the highest luciferase expression, as measured by flux, in the liver, followed by muscle (SI Appendix, Table S8). Luciferase expression was also detected in the heart, lungs, kidney, and brain. Quantitation of vector was performed for isolated organs by ddPCR specific for the luciferase gene relative to a single-copy endogenous gene, apoB. The liver showed the most copies of the luciferase gene at 0.737 copies per cell, followed by muscle (0.398 copies per cell) and heart (0.317 copies per cell) (SI Appendix, Table S8). Notably, no toxicity due to AAVHSC editing was noted for up to 6 mo postinjection, the end of the study.
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