In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models

Apr 4, 2017Molecular therapy : the journal of the American Society of Gene Therapy

Removing HIV-1 Virus DNA in Living Animals Using saCas9 and Multiple Guide RNAs

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Abstract

The quadruplex sgRNAs/saCas9 AAV-DJ/8 vector excised HIV-1 proviral DNA in multiple animal models.

The all-in-one AAV vector demonstrated improved efficiency in excising the HIV-1 genome compared to a duplex vector.
Significant reductions in viral RNA expression were observed in various organs of Tg26 mice following intravenous injection.
Systemic EcoHIV infection in acutely infected mice was reduced after treatment with the quadruplex sgRNAs/saCas9 vector.
Efficient proviral excision was confirmed in the liver, lungs, brain, and spleen using PCR genotyping.
Successful proviral excision was also detected in humanized BLT mice with chronic HIV-1 infection after a single treatment.

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CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). 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. 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.

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