Targeted Integration and High-Level Transgene Expression in AAVS1 Transgenic Mice after In Vivo HSC Transduction with HDAd5/35++ Vectors

Mol Ther. 2019 Dec 4;27(12):2195-2212. doi: 10.1016/j.ymthe.2019.08.006. Epub 2019 Aug 19.


Our goal is the development of in vivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a CRISPR/Cas9 specific to the "safe harbor" adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR + HDAd-donor vector system in AAVS1 transgenic mice using a standard ex vivo HSC gene therapy approach as well as a new in vivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after in vivo selection with low-dose O6BG/bis-chloroethylnitrosourea (BCNU). Ex vivo and in vivo HSC transduction and selection studies with HDAd-CRISPR + HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after in vivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.

Keywords: AAVS1; CRISPR/Cas9; helper-dependent adenovirus; hematopoietic stem cell; targeted integration; transgenic mice.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics*
  • Animals
  • CRISPR-Cas Systems
  • Dependovirus / genetics*
  • Female
  • Genes, Reporter
  • Genetic Therapy
  • Genetic Vectors / genetics*
  • Hematopoietic Stem Cell Mobilization
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Transduction, Genetic*
  • Transgenes / physiology*
  • Virus Integration*
  • gamma-Globins / antagonists & inhibitors
  • gamma-Globins / genetics


  • gamma-Globins