Meganucleases and Other Tools for Targeted Genome Engineering: Perspectives and Challenges for Gene Therapy

Abstract

The importance of safer approaches for gene therapy has been underscored by a series of severe adverse events (SAEs) observed in patients involved in clinical trials for Severe Combined Immune Deficiency Disease (SCID) and Chromic Granulomatous Disease (CGD). While a new generation of viral vectors is in the process of replacing the classical gamma-retrovirus – based approach, a number of strategies have emerged based on non-viral vectorization and/or targeted insertion aimed at achieving safer gene transfer. Currently, these methods display lower efficacies than viral transduction although many of them can yield more than 1% engineered cells in vitro. Nuclease-based approaches, wherein an endonuclease is used to trigger site-specific genome editing, can significantly increase the percentage of targeted cells. These methods therefore provide a real alternative to classical gene transfer as well as gene editing. However, the first endonuclease to be in clinic today is not used for gene transfer, but to inactivate a gene (CCR5) required for HIV infection. Here, we review these alternative approaches, with a special emphasis on meganucleases, a family of naturally occurring rare-cutting endonucleases, and speculate on their current and future potential. Keywords: Homing endonuclease, Zinc-finger nuclease, Recombinase, Transposons, gene transfer, protein engineering, viral vector, Severe Combined Immune Deficiency Disease, Chromic Granulomatous Disease, transposases, Streptomyces phage, human embryonic stem, pluripotent stem, chimeric antibody receptor, Zinc-finger nucleases, Activator Like Effectors, pseudo-symmetric, meganucleases, protein-protein interactions, single-strand annealing, leukemia, Adeno-Associated Virus, Haematopoietic Stem Cell, Triplex-Forming Oligonucleotides, Zinc-Finger Protein