The use of Matrigel combined with encapsulated cell technology to deliver a complement inhibitor in a mouse model of choroidal neovascularization

Abstract

Purpose: Risk for age-related macular degeneration (AMD), a slowly progressing, complex disease, is tied to an overactive complement system. Efforts are under way to develop an anticomplement-based treatment to be delivered locally or systemically. We developed an alternative pathway (AP) inhibitor fusion protein consisting of a complement receptor-2 fragment linked to the inhibitory domain of factor H (CR2-fH), which reduces the size of mouse choroidal neovascularization (CNV) when delivered locally or systemically. Specifically, we confirmed that ARPE-19 cells genetically engineered to produce CR2-fH reduce CNV lesion size when encapsulated and placed intravitreally. We extend this observation by delivering the encapsulated cells systemically in Matrigel.

Methods: ARPE-19 cells were generated to stably express CR2 or CR2-fH, microencapsulated using sodium alginate, and injected subcutaneously in Matrigel into 2-month-old C57BL/6J mice. Four weeks after implantation, CNV was induced using argon laser photocoagulation. Progression of CNV was analyzed using optical coherence tomography. Bioavailability of CR2-fH was evaluated in Matrigel plugs with immunohistochemistry, as well as in ocular tissue with dot blots. Efficacy as an AP inhibitor was confirmed with protein chemistry.

Results: An efficacious number of implanted capsules to reduce CNV was identified. Expression of the fusion protein systemically did not elicit an immune response. Bioavailability studies showed that CR2-fH was present in the RPE/choroid fractions of the treated mice, and reduced CNV-associated ocular complement activation.

Conclusions: These findings indicate that systemic production of the AP inhibitor CR2-fH can reduce CNV in the mouse model.

Figure 1

Encapsulated cell technology to deliver CR2-fH: Tool development. A: Detection of CR2-fH in the tissue surrounding the ARPE-19 cell-containing matrigel plug with immunohistochemistry using an antibody against CR2. The corresponding differential interference contrast (DIC) and fluorescence image is presented. CR2 antibody staining was negative in control tissues containing empty alginate capsules. Scale bar: 100 μm. B: Systemically produced CR2-fH could trigger an immune response. Lack of immunoglobulin G (IgG) or IgM antibody production was confirmed 1 month after capsule injection. Purified CR2-fH was run at two different concentrations and probed for the presence of CR2-fH using the anti-CR2 antibody (positive control). Identical lanes were probed with serum from experimental animals (S: CR2-fH) or control animals (S: control) at 1:50, followed by the appropriate secondary antibodies.

Figure 2

Encapsulated cell technology to deliver CR2-fH: Identification of a therapeutic dose. A: After systemic capsule delivery, CR2-fH was detectable in the RPE/choroid fraction of eyes with choroidal neovascularization (CNV) lesions. A dot blot of RPE/choroid samples with twofold dilution steps is presented documenting a dose-dependent increase in CR2 and CR2-fH delivery and binding to the tissues. B: CNV-induced complement activation in untreated animals was demonstrated by elevated levels of C3a when compared to animals with no lesions (control). C3a levels remained elevated in animals exposed to CR2, and statistically significantly reduced by CR2-fH. C: CNV sizes were reduced in animals injected systemically with ARPE-19 cells expressing CR2-fH as opposed to those expressing CR2, with an apparent efficacious dose of 1,000 capsules. Data shown are average values (± standard error of the mean [SEM]; n = 2–3 animals per condition).

Figure 3

Systemic ECT-mediated delivery of CR2-fH reduces CNV and complement activation. One month following subcutaneous injection of alginate capsules, laser choroidal neovascularization (CNV) was induced. On day 5 after injury, the lesion sizes were analyzed with optical coherence tomography (OCT; lesion size and height), and complement activation was determined using enzyme-linked immunosorbent assay (ELISA) for the C3 breakdown product C3a. A, B: CNV sizes and lesion heights were statistically significantly reduced in animals treated with ARPE-19 cells expressing CR2-fH as opposed to those expressing CR2. The gray line in the en face image can be used to pick the locations of B-scans, but are not used here; CNV lesion height is measured between BrM (black line) and its peak. C: CNV induced complement activation as demonstrated by elevated levels of C3 breakdown product C3a when compared to animals with no lesions (control), an effect that was mitigated by CR2-fH but not CR2. Data shown are average values (± standard error of the mean [SEM]; n = 5–8 animals per condition).