HIV-1 mRNA knockdown with CRISPR/CAS9 enhances neurocognitive function

• Published in: Journal of neurovirology Vol. 30; no. 1; pp. 71 - 85
• Main Authors: McLaurin, Kristen A., Li, Hailong, Khalili, Kamel, Mactutus, Charles F., Booze, Rosemarie M.
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.02.2024
• Subjects: Animals; Biomedical and Life Sciences; Biomedicine; CRISPR-Cas Systems; Gene Editing - methods; HIV Seropositivity; HIV-1 - genetics; Humans; Immunology; Infectious Diseases; Neurology; Neurosciences; Rats; RNA, Messenger - genetics; Virology; Cell culture; In situ hybridization; Gene editing; Microglia; Prepulse inhibition
• ISSN: 1355-0284; 1538-2443; 1538-2443
• DOI: 10.1007/s13365-024-01193-z

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 × 10 9 , 8.46 × 10 9 , 1.269 × 10 10 , 1.692 × 10 10 , 2.115 × 10 10 , and 2.538 × 10 10 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.