Abstract
Purpose :
Age-related macular degeneration (AMD) is the primary cause of irreversible blindness in the elderly. The current paradigm for wet AMD treatment is monthly intravitreal injection of anti-VEGF antibodies, which poses a significant economic burden to the patients. Continuous blocking of VEGF may have adverse effect in choroid capillaries and the ciliary body. We propose a gene therapy approach to drive inducible and reversible VEGF repression using a CRISPR-based system, which could avoid repetitive intravitreal injection while achieving controllable VEGF repression.
Methods :
ARPE-19 and mouse RPE cell lines were engineered to express Cas9-Krab reversibly as controlled by doxycycline (Dox), which was confirmed by Western blot analysis and immunostaining. A series of guide RNA pairs targeting human (mouse) VEGF promoter were used in screening to identify guide RNAs that can repress VEGF expression. constitutively expressed. Real-time PCR and ELISA were employed to quantify the VEGF expression before and after Dox treatment in the system. Laser injury model is being used to evaluate the efficacy of the approach in vivo.
Results :
Induction and dosage-response of Cas9-Krab expression were confirmed by Western blot analysis and immunostaining. In addition, Cas9-Krab can be reinduced in the same cells within 12 hours 7 days after the first induction. Guide RNAs have been screened to target VEGF promoter RPE cells. Up to 90% reduction of VEGF mRNA and protein expression has been achieved in the system.
Conclusions :
We have established a CRISPR-based reversible VEGF repression system, which could be used to treating retinal diseases including AMD and diabetic retinopathy. (This research has been supported by a grant from BrightFocus Foundation)
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.