Jin Hyoung Kim

Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.

RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene–specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.

Diabetic retinopathy (DR) is the leading cause of vision loss as a major complication of diabetes mellitus. The blood-retinal barrier (BRB) breakdown is a critical early event in the pathogenesis of DR. It has been known that the rennin-angiotensin system (RAS) is important in the progression of the DR via angiotensin II (Ang II), the effector of RAS. In this study, we showed that blockade of Ang II attenuates vascular endothelial growth factor (VEGF)-mediated BRB breakdown in DR. In streptozotocin-induced diabetes, retinal vascular permeability increased with upregulation of VEGF, where Ang II and its receptors were upregulated. Ang II induced VEGF expression in retinal endothelial cells accompanied by loss of tight junction proteins. However, the blockade of Ang II by perindopril, an angiotensin converting enzyme (ACE) inhibitor, inhibited upregulation of VEGF, and prevented the loss of tight junction proteins. Moreover, inhibition of Ang II by perindopril attenuated increased vascular permeability of diabetic retina accompanied by recovery of tight junction proteins in retinal vessels. Therefore, we suggest that the RAS involves in increased vascular permeability during early stage of DR, which is mediated by VEGF. Furthermore, the ACE inhibitor may have a therapeutic potential in the treatment of diabetic BRB breakdown.