Development of a C3 Humanized Rat as a New Model for Evaluating Novel C3 Inhibitors

Abstract

Introduction: C3 is central for all complement activation pathways, thus making it an attractive therapeutic target. Many C3-targeted agents are under extensive development with one already approved for clinical use. However, most, if not all, C3 inhibitors are human or nonhuman primate C3-specific, making evaluating their efficacies in vivo before a clinical trial extremely difficult and costly.

Methods: We first studied the compatibility of human C3 in the rat complement system, then developed a C3 humanized rat using the CRISPR/Cas9 technology. We thoroughly characterized the resultant human C3 humanized rats and tested the treatment efficacy of an established primate-specific C3 inhibitor in a model of complement-mediated hemolysis in the C3 humanized rats.

Results: We found that supplementing human C3 protein into the C3-deficient rat blood restored its complement activity, which was inhibited by rat factor H or compstatin, suggesting that human C3 is compatible to the rat complement system. The newly developed C3 humanized rats appeared healthy and expressed human but not rat C3 without detectable spontaneous C3 activation. More importantly, complement-mediated hemolysis in the C3 humanized rats was also inhibited by compstatin both in vitro and in vivo.

Conclusion: The successfully developed C3 humanized rats provided a much-desired rodent model to evaluate novel C3 inhibitors in vivo as potential drugs.

Fig. 1.

Human C3 restores C3 KO rat complement activity that is controlled by rat factor H and compstatin, a primate-specific C3 inhibitor. a 1 × 10⁵ rabbit red blood cells (E^rabb) were incubated with 20% wild-type (WT) or C3 KO serum, supplemented with 50 or 100 μg/mL human C3 in Mg-EGTA GVB buffer at 37°C for 20 min. The hemolysis% was evaluated and calculated as described in the Methods. b 0–3.4 μm of human or rat factor H were added into 5% C3 KO rat serum that was supplemented with 125 μg/mL human C3 together with E^rabb in the Mg-EGTA GVB buffer. Hemolysis was evaluated and calculated by OD₄₁₄ readings after 1 h of incubation at 37°C. c 0–50 µm of compstatin were added into 20% C3 KO rat serum supplemented with 50 μg/mL of human C3 together with E^rabb in the Mg-EGTA GVB buffer at 37°C for 20 min. 20% WT rat serum was used as a positive control, and 20% C3 KO rat serum was used as a negative control. d E^rabb were intravenously injected into C3 KO rats that were supplemented with 500 μg human C3 each. The rats were euthanized after 30 min, and blood was collected by cardiac puncture. Levels of free hemoglobin in plasma were evaluated by measuring OD₄₁₄. **p < 0.01.

Fig. 2.

Human C3 transcripts are detectable in multiple tissues from the hC3 KI rats. Total RNAs were isolated from the liver (a), spleen (b), and retina (c) tissues of male human C3 knock-in (hC3 KI) rats and DNase I-treated. Human C3 transcripts were amplified by PCR using cDNAs reverse-transcribed from these total RNAs as templates (cDNA). The same RNA samples after DNase I treatment but without reverse transcription were used as negative controls (RNA).

Fig. 3.

hC3 KI rats expressed human but not rat C3 and has no detectable spontaneous C3 activation. a 50 ng purified human C3 (hC3) or rat C3 (rC3) were boiled and loaded onto an SDS-PAGE gel, then detected by a polyclonal goat anti-human C3 IgG. The rat C3 beta chain showed a smaller molecular weight than its human counterpart. Using the same Western blot assay, human C3 proteins were detected in samples only from normal human serum (human) and hC3 KI rats (hC3KI). Rat C3 proteins were only detected in samples from WT rats (WT). Human C3-depleted serum (hC3-dpl) and C3 KO rat plasma (C3KO) were used as negative controls for plasma samples. b Human C3 levels in the plasma of both human C3 knock-in (hC3 KI) male (M) and female (F) rats were examined using a commercially available ELISA kit that specifically recognizes human C3 but not rat C3, per the manufacturer’s instruction. WT rat plasma (WT) was diluted at the same condition as hC3 KI rats and served as the negative control. Normal human plasma (human) was diluted for an additional 1:50 fold as compared to hC3 KI plasma and served as the positive control. c Human C3 levels in the vitreous and aqueous humor samples of male WT and hC3 KI male rats were detected using the same human C3 ELISA kit. d C3a levels in hC3 KI male rats were evaluated using a cytometric bead array-based assay. Cobra venom factor (CVF)-activated hC3 KI rat serum was used as positive controls. **p < 0.01.

Fig. 4.

hC3 KI rats process complement activity in vitro and in vivo that can be inhibited by compstatin. a 5–80% hC3 KI male rat serum or WT rat serum (WT rat) or normal human serum (human) were incubated with antibody-sensitized sheep red blood cells (E^ShA) at 37°C for 20 min in GVB⁺⁺ buffer. The hemolysis% was determined by OD₄₁₄ readings. b 5–80% hC3 KI male rat serum or WT rat serum or normal human serum or C3 KO rat serum were incubated with E^rabb for 1 h in Mg-EGTA GVB buffer. The hemolysis was evaluated by OD₄₁₄ readings. c 0–5 µM compstatin were incubated with E^rabb mixed with 50% hC3 KI male rat serum, or 20% WT rat serum, or 10% normal human serum in Mg-EGTA GVB buffer at 37°C for 1 h. The hemolysis% was determined by OD₄₁₄ readings. d 2 × 10⁹ E^rabb were injected intravenously into each hC3 KI male rats, with or without immediate treatment of 20 μm compstatin (Comp). The rats were euthanized after 60 min, and blood was collected by cardiac puncture. Levels of released hemoglobin in serum were evaluated via measuring OD₄₁₄. The baseline reading of rat serum was evaluated by collecting WT naïve rat serum (without E^rabb infusion) via cardiac puncture after euthanasia (naïve WT). *p < 0.05, **p < 0.01; ns, no significance.