Mol Ther Methods Clin Dev. 2023 Aug 28
Yaowei Guo, Junliang Chen, Wenyu Ji, Liang Xu, Yu Xie, Shu He, Chuying Lai, Kaiyu Hou, Zeru Li, Gong Chen and Zheng Wu
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AAV vector packaging AAV vector production and titration SsAAV vectors were used in this study, and all vector genomes were flanked by AAV2/9 (AAV9) or AAV2/5 (AAV5) ITRs. AAV vectors, GFAP-GFP and CAG-Flex-GFP, were constructed by PackGene Biotech. The versions of the GFAP promoter used in this study included GFAP2.2 (2.2-kb gfa2 promoter), GFAP1.6 (1.6-kb GFAP promoter), and GFAP681 (681-bp gfaABC1D promoter). Recombinant AAV9 and AAV5 were produced by PackGene Biotech Request Quote

Research Field: CNS

Keywords: AAV, astrocytes, immune response, BBB disruption, lymphocyte infiltration, neuronal toxicity

AAV Serotype: AAV5, AAV9

Dose: 10E8, 10E10

Routes of Administration: Stereotaxic viral injection, 0.1–1 mL of AAV was injected into the brain at a speed of 100 nL/min

Targeted organ: brain

Animal or cell line strain: mouse


The brain is often described as an “immune-privileged” organ due to the presence of the blood-brain-barrier (BBB), which limits the entry of immune cells. In general, intracranial injection of adeno-associated virus (AAV) is considered a relatively safe procedure. In this study, we discovered that AAV, a popular engineered viral vector for gene therapy, can disrupt the BBB and induce immune cell infiltration in a titer-dependent manner. First, our bulk RNA sequencing data revealed that injection of high-titer AAV significantly upregulated many genes involved in disrupting BBB integrity and antiviral adaptive immune responses. By using histologic analysis, we further demonstrated that the biological structure of the BBB was severely disrupted in the adult mouse brain. Meanwhile, we noticed abnormal leakage of blood components, including immune cells, within the brain parenchyma of high-titer AAV injected areas. Moreover, we identified that the majority of infiltrated immune cells were cytotoxic T lymphocytes (CTLs), which resulted in a massive loss of neurons at the site of AAV injection. In addition, antagonizing CTL function by administering antibodies significantly reduced neuronal toxicity induced by high-titer AAV. Collectively, our findings underscore potential severe side effects of intracranial injection of high-titer AAV, which might compromise proper data interpretation if unaware of.

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