Abstract

Mixed glia are infiltrated with HIV-1 virus early in the course of infection leading to the development of a persistent viral reservoir in the central nervous system. Modification of the HIV-1 genome using gene editing techniques, including CRISPR/Cas9, has shown great promise towards eliminating HIV-1 viral reservoirs; whether these techniques are capable of removing HIV-1 viral proteins from mixed glia, however, has not been systematically evaluated. Herein, the efficacy of adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing for eliminating HIV-1 messenger RNA (mRNA) from cortical mixed glia was evaluated in vitro and in vivo. In vitro, a within-subjects experimental design was utilized to treat mixed glia isolated from neonatal HIV-1 transgenic (Tg) rats with varying doses (0, 0.9, 1.8, 2.7, 3.6, 4.5, or 5.4 µL corresponding to a physical titer of 0, 4.23 × 10⁹, 8.46 × 10⁹, 1.269 × 10¹⁰, 1.692 × 10¹⁰, 2.115 × 10¹⁰, and 2.538 × 10¹⁰ gc/µL) of CRISPR/Cas9 for 72 h. Dose-dependent decreases in the number of HIV-1 mRNA, quantified using an innovative in situ hybridization technique, were observed in a subset (i.e., n = 5 out of 8) of primary mixed glia. In vivo, HIV-1 Tg rats were retro-orbitally inoculated with CRISPR/Cas9 for two weeks, whereby treatment resulted in profound excision (i.e., approximately 53.2%) of HIV-1 mRNA from the medial prefrontal cortex. Given incomplete excision of the HIV-1 viral genome, the clinical relevance of HIV-1 mRNA knockdown for eliminating neurocognitive impairments was evaluated via examination of temporal processing, a putative neurobehavioral mechanism underlying HIV-1-associated neurocognitive disorders (HAND). Indeed, treatment with CRISPR/Cas9 protractedly, albeit not permanently, restored the developmental trajectory of temporal processing. Proof-of-concept studies, therefore, support the susceptibility of mixed glia to gene editing and the potential of CRISPR/Cas9 to serve as a novel therapeutic strategy for HAND, even in the absence of full viral eradication.

中文翻译：

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使用 CRISPR/CAS9 敲低 HIV-1 mRNA 可增强神经认知功能

摘要

在感染过程的早期，混合神经胶质细胞被 HIV-1 病毒浸润，导致中枢神经系统中形成持久的病毒库。使用基因编辑技术（包括 CRISPR/Cas9）对 HIV-1 基因组进行修饰，在消除 HIV-1 病毒库方面显示出了巨大的希望；然而，这些技术是否能够从混合神经胶质细胞中去除 HIV-1 病毒蛋白尚未得到系统评估。在此，我们在体外和体内评估了腺相关病毒 9 (AAV9)-CRISPR/Cas9 基因编辑从皮质混合胶质细胞中消除 HIV-1 信使 RNA (mRNA) 的功效。在体外，采用受试者内实验设计，以不同剂量（对应于物理滴度的 0、0.9、1.8、2.7、3.6、4.5 或 5.4 µL）治疗从新生 HIV-1 转基因 (Tg) 大鼠中分离的混合神经胶质细胞。 0、4.23 × 10 ⁹、8.46 × 10 ⁹、1.269 × 10 ¹⁰、1.692 × 10 ¹⁰、2.115 × 10 ¹⁰和 2.538 × 10 ¹⁰ gc/μL) CRISPR/Cas9 72 小时。在原 代混合神经胶质细胞的子集（即，8 个中，n = 5 个）中观察到 HIV-1 mRNA 数量的剂量依赖性减少，使用创新的原位杂交技术进行量化。在体内，HIV-1 Tg 大鼠眼眶后接种 CRISPR/Cas9 两周，治疗导致内侧前额叶皮质中 HIV-1 mRNA 的深度切除（即约 53.2%）。鉴于 HIV-1 病毒基因组的不完全切除，通过检查时间处理（HIV-1 相关神经认知障碍 (HAND) 的假定神经行为机制）来评估 HIV-1 mRNA 敲低对于消除神经认知障碍的临床相关性。事实上，尽管不是永久的，但长期使用 CRISPR/Cas9 治疗可以恢复时间处理的发育轨迹。因此，概念验证研究支持混合神经胶质细胞对基因编辑的敏感性，以及 CRISPR/Cas9 作为 HAND 新型治疗策略的潜力，即使在没有完全根除病毒的情况下也是如此。