bioRxiv. 2020 Sep 3
Gong Chen, Wen Li, Zongqin Xiang, Liang Xu, Minhui Liu, Qingsong Wang and Wenliang Lei
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AAV vector packaging AAV serotype 9 (AAV9) and 5 (AAV5) were produced by PackGene® Biotech, LLC, purified through iodixanol gradient ultracentrifuge and subsequent concentration. Request Quote

Research Field: CNS

AAV Serotype: AAV9 and AAV5

Dose: 7 days later, 1 μL of virus mixture AAV9 hGFAP::Cre (1×10^10 GC/ml) and FLEX-CAG::mCherry (1×10^12 GC/ml) was injected at the same coordinates.

Routes of Administration: 1 μL of ET-1 (1 μg/μL dissolved in PBS) was injected at the following coordinate: +0.2 mm anterior-posterior (AP), ± 1.5 mm medial-lateral (ML), 1.2 mm dorsal-lateral (DV) at the speed of 100 nl/min. After injection, the pipette was kept in place for about 10 minutes and then slowly withdrawn. 7 days later, 1 μL of virus mixture AAV9 hGFAP::Cre (1×10^10 GC/ml) and FLEX-CAG::mCherry (1×10^12 GC/ml) was injected at the same coordinates.

Targeted organ: brain

Animal or cell line strain: adult WT C56BL/6J mice


Regenerating functional new neurons in the adult mammalian central nervous system (CNS) has been proven to be very challenging due to the inability of neurons to divide and repopulate themselves after neuronal loss. In contrast, glial cells in the CNS can divide and repopulate themselves under injury or disease conditions. Therefore, many groups around the world have been able to utilize internal glial cells to directly convert them into neurons for neural repair. We have previously demonstrated that ectopic expression of NeuroD1 in dividing glial cells can directly convert reactive glial cells into neurons. However, Wang et al. recently posted an article in bioRxiv challenging the entire field of in vivo glia-to-neuron conversion after using one single highly toxic dose of AAV (2×1013 gc/ml, 1 μl) in the mouse cortex, producing artifacts that are very difficult to interpret. We present data here that reducing AAV dosage to safe level will avoid artifacts caused by toxic dosage. We also demonstrate with Aldh1l1-CreERT2 and Ai14 reporter mice that lineage-traced astrocytes can be successfully converted into NeuN+ neurons after infected by AAV5 GFAP::NeuroD1. Retroviral expression of NeuroD1 further confirms our previous findings that dividing glial cells can be converted into neurons. Together, the incidence of Wang et al. sends an alarming signal to the entire in vivo reprogramming field that the dosage of viral vectors is a critical factor to consider when designing proper experiments. For AAV, we recommend a relatively safe dose of 1×1010 – 1×1012 gc/ml (~1 μl) in the rodent brain for cell conversion experiments addressing basic science questions. For therapeutic purpose under injury or diseased conditions, AAV dosage needs to be adjusted through a series of dose finding experiments. Moreover, we recommend that the AAV results are further verified with retroviruses that mainly express transgenes in dividing glial cells in order to draw solid conclusions.

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