Abstract
Purpose :
Variants of Complement factor H (CFH) and its alternative splice form, FHL-1, are major genetic risk factors for AMD but the relative role/contribution of each form to AMD development is unknown. Our lab has developed mouse models of AMD combining major risk factors of human disease, including complement dysregulation through CFH insufficiency, advanced aging, and a high-fat, high-cholesterol (HFC) diet, simulating a Western diet. Using AAVs expressing FHL-1 to examine the function of FHL-1 in Cfh knock out (Cfh-/-) mice, we tested the hypothesis that FHL-1 directly contributes to complement regulation in vivo and modulates ocular phenotypes in one of our models.
Methods :
AAV vectors were designed for systemic expression of FHL-1 with a triple Myc tag (FHL-1-3xMyc), using a liver-specific thyroxine-binding globulin (TBG) promoter and delivered by tail vein injection in Cfh-/- mice. Effectiveness of the vectors was tested in young Cfh-/- mice. FHL-1-3xMyc expression was determined by Western blot, and complement activity was assessed by Western blot of complement proteins C3, FB, and C5. The effect of FHL-1-3xMyc on ocular phenotypes was assessed in aged (>90 weeks) Cfh-/- mice fed a high fat, cholesterol-enriched (HFC) diet (Cfh-/-~HFC). Visual function was measured by electroretinography (ERG).
Results :
Cfh-/- mice injected with the AAV constructs had detectable levels of FHL-1-3xMyc and intact C5 in their plasma. FHL-1-3xMyc was also detectable in perfused eyecups of these mice, demonstrating retention of circulating protein. An increase in uncleaved C5 was detectable in Cfh-/- mice receiving the virus. Visual function of Cfh-/-~HFC was affected by expression of FHL-1-3xMyc, resulting in an ERG b-wave deficit normally absent in this genotype after diet consumption.
Conclusions :
These studies establish FHL-1-3xMyc expressed by the liver following viral delivery can affect circulating levels of complement and ocular health as evident by increased plasma C5 levels and attenuated ERG responses. Development of a visual deficit in mice receiving the liver-targeting AAVs expressing FHL-1-3xMyc supports a role of circulating complement in the development of ocular pathologies in our mouse models. These results support a role of systemic complement in AMD pathobiology and further studies of novel AMD therapies targeting the complement system.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.