They demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an allin-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9).<\/p>\n
The quadruplex sgRNAs\/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem\/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs\/saCas9 AAV-DJ\/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs\/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs\/saCas9 AAV-DJ\/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vector induced efficient proviral excision, as determined by PCR genotyping in the liver, lungs, brain, and spleen.<\/p>\n
Finally, in humanized bone marrow\/liver\/thymus (BLT) mice with chronic HIV-1 infection, successful proviral excision was detected by PCR genotyping in the spleen, lungs, heart, colon, and brain after a single intravenous injection of quadruplex sgRNAs\/saCas9 AAV-DJ\/8. In conclusion, in vivo excision of HIV-1 proviral DNA by sgRNAs\/saCas9 in solid tissues\/organs can be achieved via AAV delivery, a significant step toward human clinical trials.<\/p>\n