Making gene editing a therapeutic reality

Abstract

This review discusses current bottlenecks in making CRISPR-Cas9-mediated genome editing a therapeutic reality and it outlines recent strategies that aim to overcome these hurdles as well as the scope of current clinical trials that pioneer the medical translation of CRISPR-Cas9. Additionally, this review outlines the specifics of disease-modifying gene editing in recessive versus dominant genetic diseases with the focus on genetic myopathies that are exemplified by Duchenne muscular dystrophy and myotonic dystrophies.

Keywords: AAV; Alpha1 Antitrypsin deficiency; CRISPR; Clinical trials; DMD; DNA damage; HDR; Myotonic Dystrophy; NHEJ; nanoparticles.

Figure 1.. Roadblocks for clinical translation of CRISPR-Cas9.

(1) Circulating anti-AAV antibodies will cause an immune response and eliminate AAV nanoparticles carrying Cas9 gRNA. (2) Owing to the imprecision of current delivery systems, there is dilution of the therapeutic CRISPR-Cas9 by less relevant cells and tissues in the body. (3) Many cells in the body, including quiescent stem cells and post-mitotic differentiated cells, have poor DNA repair, thus making CRISPR-Cas9 therapies insufficient for correcting mutated genes with high-enough efficiency. (4) CRISPR-Cas9 causes unintended off-target DNA damage which not only can lead to gene inactivation or ectopic activation but also activates p53, thus promoting cell apoptosis, particularly when DNA repair is intrinsically inefficient. (5) Proliferative cells with CRISPR-Cas9-induced damage might undergo oncogenic transformations. Despite these obstacles, CRISPR-Cas9 is moving forward in clinical trials. Several ways to overcome the bottlenecks in clinical translation of CRISPR-Cas9 are being pursued and are described in this perspective. AAV, adenoviral-associated virus; CRISPR, clustered regularly interspaced short palindromic repeats.

Figure 2.. Specifics of CRISPR-Cas9 NHEJ approach to DMD.

Dystrophin-negative myofibers have inactivating dystrophin gene mutation(s), resulting in the absence of dystrophin protein. Delivery of CRISPR-Cas9 (AAV-Cas9 and AAV-gRNAs) (1) leads to editing of the mutated exon (that is, removing a premature stop codon, red box) in rare muscle precursor cells, myoblasts, which can divide, migrate, and fuse with a number of myofibers (2). Many non-dividing cells in the muscle, including the quiescent MuSCs (muscle stem cells) and myofibers, are not efficiently edited by CRISPR-Cas9, and other muscle-resident cells, such as fibroblasts, might be edited. Truncated but partially functional dystrophin protein is delivered to clusters of myofibers (green outlines) through fusion events with the rare CRISPR-Cas9-edited myoblasts, where 1,000s copies of dystrophin mRNAs might be produced by a single corrected myonucleus (3). AAV, adenoviral-associated virus; CRISPR, clustered regularly interspaced short palindromic repeats; DMD, Duchenne muscular dystrophy; NHEJ, non-homologous end joining.