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

Mol Ther. 2017 May 3;25(5):1168-1186. doi: 10.1016/j.ymthe.2017.03.012. Epub 2017 Mar 30.

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.

Keywords: AAV; Cas9; HIV; Tg26 mice; animal models; gRNA; gene therapy; genome editing; humanized BLT mice; viral latency.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Dependovirus / genetics
  • Dependovirus / metabolism
  • Disease Models, Animal
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Gene Editing / methods
  • Genetic Therapy / methods*
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Genome, Viral*
  • HIV Infections / pathology
  • HIV Infections / therapy*
  • HIV Infections / virology
  • HIV Long Terminal Repeat
  • HIV-1 / genetics*
  • HIV-1 / metabolism
  • Humans
  • Mice
  • Mice, Transgenic
  • Oligonucleotides / genetics
  • Oligonucleotides / metabolism
  • Proviruses / genetics*
  • Proviruses / metabolism
  • RNA, Guide / genetics*
  • RNA, Guide / metabolism
  • Staphylococcus aureus / chemistry
  • Staphylococcus aureus / enzymology
  • gag Gene Products, Human Immunodeficiency Virus / genetics
  • gag Gene Products, Human Immunodeficiency Virus / metabolism
  • pol Gene Products, Human Immunodeficiency Virus / genetics
  • pol Gene Products, Human Immunodeficiency Virus / metabolism

Substances

  • Bacterial Proteins
  • Oligonucleotides
  • RNA, Guide
  • gag Gene Products, Human Immunodeficiency Virus
  • pol Gene Products, Human Immunodeficiency Virus
  • Endonucleases