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

Abstract

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.

Excision of Latent HIV-1 from Infected Cells In Vivo: An Important Step Forward

“…8 Complementary evidence also suggests that a combinatorial approach targeting two or more regions of the HIV genome that can facilitate higher proviral mutagenesis could circumvent viral escape. In the present study, Yin et al 2 showed that combining sgRNAs targeting LTR and structural proteins resulted in higher levels of both small indels and excision of HIV segments between target sites, resulting in higher overall disruption efficiency. These properties will play crucial roles as HIV genome editing progresses toward clinical trials by minimizing concerns about HIV diversity and the emergence of resistance.…”

“…In this issue of Molecular Therapy, Yin and colleagues take an important step in moving this approach to in vivo application by investigating the feasibility of Cas9-mediated provirus excision in three murine models of HIV-1 infection. 2 Their ability to deliver an all-in-one adeno-associated virus (AAV) vector containing multiple singleguide RNAs (sgRNAs) in tandem with Staphylococcus aureus (sa)Cas9 and to detect HIV-1provirus excision in multiple tissues and organs in models, including bone marrow-liver-thymus (BLT) mice with chronic HIV-1 infection, represents an exciting milestone in moving the field toward human trials.…”

“…Yin et al 2 demonstrated that an oversized cargo of 5.7 kb, encoding four multiplexed sgRNAs along with saCas9, can be efficiently packaged into AAV-DJ and AAV-DJ/8 vectors and delivered in vivo. AAV was a logical choice of gene transfer vector for this application.…”

“…Each cell of the Tg26 transgenic mouse contains a tandem repeat of 10-20 copies of a replication incompetent 7.4 kb pNL4-3 proviral construct. Yin et al 2 administered multiplexed saCas9 AAV-DJ or AAV-DJ/8 through tail vein injection and demonstrated efficient expression of saCas9 in various solid tissues and organs throughout the mice as early as 1 week. 4 weeks later, the researchers also observed fragmentation of the integrated HIV provirus by PCR genotyping in various tissues, including brain, heart, liver, lung, kidney, and spleen.…”

Future Perspectives for Cancer Therapy Using the CRISPR Genome Editing Technology

“…Yin et al recently revealed that the CRISPR-Cas9 technology can be used to excise the HIV-1 DNA from the genome of a "humanized" mouse that carries HIV-1-infected human lymphocytes [43]. The authors also demonstrated that the gene-editing apparatus can be engineered to carry a set of multiple guide RNAs, all designed to efficiently excise integrated HIV-1 DNA from the host cell genome, a strategy that might well overcome a potential escape of mutated HIV-1 viruses.…”