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.

Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

“…Zinc-finger nucleases, meganucleases (MNs), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) have emerged during the past decade as efficient tools for genome editing in many organisms 24,25 . These molecular scissors are able to introduce a targeted DNA double-strand break that is repaired by one of two major mechanisms: non-homologous end-joining, which leads to gene disruption by inducing mutations at the break site, or homologous recombination, which drives gene insertion or gene replacement using exogenous DNA templates with homology to the targeted locus (Fig.…”

Genome-Editing Technologies: Principles and Applications

“…These enzymes have been reviewed at length elsewhere (Silva et al 2011;Stoddard 2014) but, briefly, members of the LAGLIDADG family of endonucleasesso named for the conserved amino acid motif present within these enzymes that interacts with DNA-are a collection of naturally occurring enzymes that recognize and cleave long DNA sequences (14 -40 bps) (Fig. 1).…”

Genome-Edited T Cell Therapies

“…This cleavage event initiates the transposition of the endonuclease mobile sequence into the cut site, a mechanism termed “homing” [14]. Site-specific meganucleases have challenging design criteria owing to the single-chained recognition and cleavage domains, although successful modification has been described for a number of applications [15–17]. In the context of gene therapy, meganucleases have for example been modeled for targeted recombination and correction of the RAG1 gene associated with severe combined immunodeficiency (SCID) [18] in hematopoietic stem cells (HSCs) and the XPC gene associated with Xeroderma Pigmentosum in skin cells [19].…”