Debugging the genetic code: non-viral in vivo delivery of therapeutic genome editing technologies

Abstract

Efforts to precisely correct genomic mutations that underlie hereditary diseases for therapeutic benefit have advanced alongside the emergence and improvement of genome engineering technologies. These methods can be divided into two main classes: active nucleasebased platforms including the popular CRISPR/Cas9 system and oligo/polynucleotide strategies including triplex-forming oligonucleotides (TFOs), such as peptide nucleic acids (PNAs). These technologies have been successful in cell culture and in animals, but important challenges remain before these tools can be translated into the clinic; they must be effectively delivered to and taken up by specific cell types of interest, achieve correction levels in target cells that significantly ameliorate the disease phenotype, and demonstrate minimal off-target and toxicity effects. Here we review and compare the current strategies and non-viral delivery methods, mainly lipid and polymeric vehicles, proposed for genome editing of inherited disorders with a focus on in vivo delivery and efficacy. While the path to a safe and effective medical treatment may be arduous, the future outlook of therapeutic genome editing remains promising as long as precise technologies can be combined with efficient delivery.

Keywords: CRISPR/Cas9; non-viral delivery; nuclease-mediated genome editing; oligonucleotide-mediated genome editing; peptide nucleic acid; therapeutic genome editing.

Figure 1.

Schematic summarizing non-viral strategies to deliver genome editing agents in vivo.

Figure 2.. Schematic of gene editing machinery components that need to be encapsulated in delivery vehicles and their editing mechanisms.

The components required for nuclease-mediated genome editing are shown on the left (CRISPR/Cas9 system), and the components required for TFO-mediated genome editing are shown on the right (PNA system). Approximate sizes of each of the components are indicated. Shown below the editing system components are the mechanisms of site-specific gene correction for each type of editing agent.