Figure 5.

Pharmacological intervention using genetic methods.A, gene augmentation therapy for patients with a defective RPE65 gene (reviewed in (185)). The RPE65 gene is delivered by subretinal injection using an adeno-associated viral (AAV) vector that lacks the capacity for replication. The appropriate tropism of the vector (serotype 2) allows specific transduction of the RPE cells, in which the RPE65 transgene remains episomal to the host DNA. Luxturna (Spark Therapeutics) has been approved as a therapeutic agent to treat Leber congenital amaurosis (LCA) caused by inactivating mutations in RPE65. B, genome engineering using the CRISPR-Cas9 system. The bacterial-derived Cas9 nuclease (shaded in yellow) complexed with a single-guide RNA (sgRNA, green) recognizes and binds to the target DNA that is complementary to the sequence of sgRNA (modified from (186)). Cas9 cleaves double-stranded DNA, which can be repaired in the error-prone nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. However, NHEJ can result in random indel mutations at the site of the junction and can result in frameshifts or a premature stop codon, resulting in gene knockout. In the HDR pathway, the DNA double-stranded breaks can be repaired with a homologous sequence template. C, CRISPR-Guided DNA Base Editors. The adenine base editor is constructed in a manner to target DNA by a Cas9 nickase (nCas9), with an adenosine deaminase (ecTadA), which deaminates a target adenosine (A) to inosine (I). Because inosine is recognized as guanosine (G) by the cellular machinery, the adenine base editor leads to conversion of the original A·T base pair to a G·C base pair by a DNA repair process (modified from (187)). Similarly, a cytidine base can be modified to uridine (recognized as thymidine), resulting in a G to A transition.