Abstract
Purpose :
Excessive activation of complement causes many diseases such as paroxysmal nocturnal hemoglobinuria (PNH) and age-related macular degeneration (AMD). C3 is an attractive therapeutic target and many C3-targeted agents are under extensive development with one already approved for treating PNH and demonstrated efficacy for treating AMD. However, most, if not all C3 inhibitors are primate C3-specific, making evaluating their therapeutic potentials in vivo before a clinical trial extremely difficult and costly. Given the similarities between human and rat complement systems, we aimed to develop a C3 humanized rat to facilitate the development of novel C3-targeted therapeutics.
Methods :
Human C3 compatibility with the rat complement system was examined both in vitro and in vivo using the C3 knockout (KO) rats that we previously generated . Compstatin, a known primate-specific C3 inhibitor, was also used to demonstrate the hemolytic activity of the human C3-supplemented C3 KO rat sera. After this, a human C3 expression construct was knocked into the exon 1 of the rat C3 gene using the CRISPR/Cas 9 technology to generate the C3 humanized rats. The presence of human but not rat C3 in the blood from these resultant rats was verified by ELISA and western blot. Complement activity of the C3 humanized rat was assessed both in vitro and in vivo via complement-mediated hemolytic assays.
Results :
Complement activity in C3 KO rat was restored by supplementing human C3 both in vitro and in vivo, and it can be inhibited by compstatin in a concentration-dependent manner, suggesting that human C3 is compatible with the rat complement system and that this hybrid complement system could be used to evaluate primate-specific C3 inhibitors. A founder rat was identified after CRISPR/Cas9-mediated human C3 knock-in event and used to generate the desired C3 humanized rat. The resultant rats expressed human but not rat C3 in the blood and had complement-mediated hemolytic activities that can also be inhibited by compstatin.
Conclusions :
Human C3 is compatible with the rat complement system. A C3 humanized rat is successfully developed in which only human but not rat C3 is expressed. This C3 humanized rat provides an invaluable preclinical model for the development of novel human C3-targeted therapeutics without the requirement of costly non-human primates.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.