Therapeutic genome editing: prospects and challenges

Abstract

Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease-based therapies as well as future prospects and challenges.

Figure 1. Types of Therapeutic Genome Modifications

The specific type of genome editing therapy depends on the nature of the mutation causing disease. a, In gene disruption, the pathogenic function of a protein is silenced by targeting the locus with NHEJ. Formation of indels on the gene of interest often result in frameshift mutations that create premature stop codons and a non-functional protein product, or non-sense mediated decay of transcripts, suppressing gene function. c, HDR gene correction can be used to correct a deleterious mutation. A DSB is induced near the mutation site in the presence of an exogenously provided, corrective HDR template. HDR repair of the break site with the exogenous template corrects the mutation, restoring gene function. d, An alternative to gene correction is gene addition. This introduces a therapeutic transgene into either the native or a non-native locus in the genome. A DSB is induced at the desired locus and an HDR template containing homology to the break site, a promoter, a transgene and a polyadenylation (polyA) sequence is introduced to the nucleus. HDR repair recovers gene function in the target locus albeit without true physiological control over gene expression. b, In NHEJ gene correction two DSBs targeted to both sides of a pathogenic expansion or insertion may be resolved by NHEJ, causing a deletion of the intervening sequences to mediate therapy. This form of treatment would require multiplexed targeting of disease causing mutations.

Figure 2. Factors Influencing Therapeutic Efficacy

For a genome editing therapy to be efficacious, enough cells carrying the desired genome modification must exist in a tissue to reverse disease. If editing is efficient, treatment will create a population of cells carrying the desired genomic modification (depicted in pink). Depending on whether the editing event creates a fitness change in target cells, edited cells will proportionally increase, or decrease relative to unedited cells (depicted in brown) over time in tissues. Proportionally high levels of cells carrying therapeutic genome modifications in a disease-affected tissue are likely to result in a therapeutic effect. However, if low levels of a secreted gene product are needed to reverse disease, then successfully editing a small number of cells may be therapeutically efficacious.

Figure 3. Ex Vivo vs. In Vivo Editing Therapy

In ex vivo editing therapy cells are removed from a patient, edited and then re-engrafted (top panel). For this mode of therapy to be successful, target cells must be capable of survival outside the body and homing back to target tissues post-transplantation. In vivo therapy involves genome editing of cells in situ (bottom panels). For in vivo systemic therapy, delivery agents that are relatively agnostic to cell identity or state would be used to effect editing in a wide range of tissue types. For example systemic delivery of AAV serotype 8 vectors has been used in preclinical models to target liver tissue with high efficiency. Alternatively, in vivo therapy, may also be achieved through local injection of viral vectors to the affected tissue, such as the eye, brain, or muscle.