ABSTRACT
Abstract .................................................................................................................. 160 6.1 Genome Engineering in Stem Cells: Overview ............................................ 160
6.1.1 Target Cells ....................................................................................... 160 6.1.2 Site-Specific DNA Breaks Mediated by Endonucleases .................. 160 6.1.3 Gene Targeting by Homology Directed Repair
Involving Donor Vectors ................................................................... 161 6.2 Adenovirus Vectors for Genome Editing-Advantages ............................... 161
6.2.1 Gene Delivery to Nondividing Cells ................................................ 161 6.2.2 Non-Integrating Nature of Most Ad Serotypes ................................ 162 6.2.3 Insert Capacity .................................................................................. 162 6.2.4 Capsid Modification to Improve Tropism to Target Cells ................ 163 6.2.5 In Vivo Application ........................................................................... 164 6.2.6 Cost of Vector Manufacturing .......................................................... 166
6.3 Adenovirus Vectors for Genome Editing-Disadvantages .......................... 166 6.3.1 Cytotoxicity of First-Generation Vectors in Stem Cells ................... 166 6.3.2 Loss of EN Expression Cassettes from Viral Genomes
During Ad Vector Production ........................................................... 166 6.3.3 Cytotoxicity Associated with Extended EN Expression .................. 167
6.4 Adenovirus Vectors for Genome Editing-Examples .................................. 170 6.4.1 EN-Based Antiviral Therapies ......................................................... 170 6.4.2 Gene Targeting in Cell Lines and Immortalized Primary Cells ...... 171 6.4.3 Genome Editing in iPS Cells to Introduce or Correct Disease
Mutations .......................................................................................... 172 6.4.4 Genome Editing in HSCs ................................................................. 172 6.4.5 Genome Editing in MSCs ................................................................. 173 6.4.6 In Vivo Genome Editing ................................................................... 173
Acknowledgments .................................................................................................. 175 References .............................................................................................................. 175
We discuss advantages and disadvantages of using adenovirus vectors (Ads) expressing engineered endonucleases (ENs) for targeted genome engineering in vitro and in vivo, in mice. For in vitro application of Ad-ENs we focus on hematopoietic stem cells, mesenchymal stem cells, and induced pluripotent stem cells. Among the advantages of using Ad vectors for EN delivery are the ability to transduce nondividing cells, the episomal nature of Ad genomes, the easiness to modify vector tropism, and the relatively low cost of vector manufacturing, which is particularly relevant for in vivo genome editing. Disadvantages include toxicity to stem cells and immunogenicity of first-generation vectors; problems that can largely be circumvented by helper-dependent Ad vectors. Problems specific to EN-expressing Ad vectors are the risk of vector genome rearrangements and loss of EN expression during vector production, cyto-/genotoxicity associated with extended EN expression from episomal Ad vector genomes in slow or nonproliferating cells, and immune responses against ENs in vivo. We review examples for in vitro and in vivo Ad-EN-mediated genome editing and conclude that more studies on the efficacy and the safety (specifically after in vivo Ad-EN application) are required to assess the utility of this vector system for genome editing in humans.
