Abstract
Purpose :
Age-related macular degeneration (AMD), a leading cause of severe vision loss in people over 50, presents in two forms: dry and wet AMD. The risk of dry AMD is known to increase with overactivation of the alternative pathway (AP) of the complement system, a process dependent on the proteolytic cleavage of factor B (CFB). This study aims to investigate the impact of liver knockdown CFB on ocular CFB levels.
Methods :
C57BL/6J mice or double humanized C3/CFB mice (Regeneron Pharmaceuticals Inc) were used in the current study. Guide RNAs targeting mouse or human CFB were encapsulated in lipid nanoparticles along with CRISPR/Cas9 and administered intravenously to C57BL/6J mice or double humanized C3/CFB mice. Plasma samples were collected pre- and post-injection. The mRNA levels were quantified using quantitative polymerase chain reaction (qPCR), while mouse Cfb protein levels were detected using Western blot (WB). Human CFB levels were determined using ELISA.
Results :
In C57BL/6J mice (non treated n= 6, CRISPR treated n= 8), WB revealed a 90% reduction in plasma Cfb and over 70% knockdown in ocular Cfb. qPCR revealed more than 80% knockdown in liver Cfb mRNA. In double humanized C3/CFB mice, (non treated n= 5, CRISPR treated n= 5), ELISA detected a 62% reduction in liver, an 83% reduction in plasma CFB and a 45% reduction in ocular CFB. Strong correlation was noted between eye, serum and liver p<0.05.
Conclusions :
The use of CRISPR/Cas9 enabled successful knockdown of CFB in the liver and serum, which corresponded with decreased ocular CFB levels. This suggests that ocular CFB is primarily derived from the bloodstream rather than local production in the eye. These findings indicate systemic targeting of CFB as an attractive approach for treating ocular complement diseases such as dry AMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.