Retrovirus-Mediated Over-Expression of Decay-Accelerating Factor Rescues Crry-Deficient Erythrocytes from Acute Alternative Pathway Complement Attack

Abstract

Decay-accelerating factor (DAF) and complement receptor 1-related gene/protein y (Crry) are two membrane-bound complement regulators on murine erythrocytes that inhibit C3/C5 convertases. Previously, we found that Crry- but not DAF-deficient erythrocytes were susceptible to alternative pathway complement-mediated elimination in vivo. To determine whether it is a unique activity or a higher level expression of Crry makes it indispensable on murine erythrocytes, we over-expressed DAF on Crry-deficient (Crry−/−) erythrocytes by retroviral vector-mediated DAF gene transduction of bone marrow stem cells. DAF retrovirus-transduced erythrocytes expressed 846 ± 127 DAF molecules/cell (DAFhigh) compared with 249 ± 94 DAF molecules/cell (DAFlow) and 774 ± 135 Crry molecules/cell on control mouse erythrocytes. DAFhigh-Crry−/− erythrocytes were significantly more resistant than either DAFlow-Crry−/−, DAF−/− -Crry+/+ or wild-type erythrocytes to classical pathway complement-mediated C3 deposition in vitro. Furthermore, increased DAF expression rescued Crry−/− erythrocytes from acute alternative pathway complement attack in vivo. Notably, long term monitoring revealed that DAFhigh-Crry−/− erythrocytes were still more susceptible than wild-type erythrocytes to complement-mediated elimination as they had a shorter half-life in complement-sufficient mice but survived equally well in complement-deficient mice. These results suggest that both a high level expression and a more potent anti-alternative pathway complement activity of Crry contributed to its indispensable role on murine erythrocytes. Additionally, they demonstrate the feasibility of using stem cell gene therapy to correct membrane complement regulator deficiency on blood cells in vivo.

Deficiency of decay-accelerating factor and complement receptor 1–related gene/protein y on murine platelets leads to complement-dependent clearance by the macrophage phagocytic receptor CRIg

“…To exclude the possibility that clodronate-liposomes may affect systemic complement activity, sera from the treated mice were analyzed using an in vitro zymosaninduced complement activation assay. 27 Neither clodronate-liposomes nor PBS-liposomes were found to affect systemic complement activity (data not shown).…”

“…Production of Crry-encoding retrovirus and transduction of mouse bone marrow stem cells were carried as described previously. 27 …”

“…The number of DAF, Crry, or CD59a molecules per platelet was estimated based on the platelet mean fluorescence intensity (MFI) and a standard curve of MFI generated by a set of 4 precalibrated PE-labeled fluorescent beads. 27,33 …”

“…Assessment of erythrocyte half-life in vivo was carried out as previously described. 18,19,27 In vitro complement deposition assays Washed platelets and erythrocytes (10 7 in 100 L) were incubated with mouse plasma/serum (at 10%, 20%, 40%, and 80%) in modified Tyrode buffer at 37°C for 20 or 60 minutes. After incubation, cells were washed 3 times with PBS and stained with biotinylated goat anti-mouse C3 and APC-conjugated streptavidin.…”

“…27,33 Briefly, platelets were stained with PE-labeled primary or secondary mAbs with known antibody/PE molecular ratios. The number of DAF, Crry, or CD59a molecules per platelet was estimated based on the platelet mean fluorescence intensity (MFI) and a standard curve of MFI generated by a set of 4 precalibrated PE-labeled fluorescent beads.…”