Abstract
Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).
Highlights
Age-related macular degeneration (AMD) is the most common cause of blindness amongst the elderly in the industrialised world, affecting approximately 36 to 40Peter J
The C3 alpha chain is degraded by Complement Factor I (CFI) into iC3b which can be detected by immunoblotting as a reduction of the alpha chain at 116 kDa and appearance of the two iC3b breakdown bands at 68 and 43 kDa. 20 μL of dissection ‘Wash’ sample collected during dissection of eyes was incubated with 1 μg purified C3b (Complement Technology Inc.) and 0.5 μg purified Complement Factor H (CFH) (Complement Technology Inc.) for 1 hour at 37 °C
The 1752 bp CFI sequence was placed under the control of the ubiquitous CAG promoter, followed by a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a bovine growth hormone poly-A site with AAV2 inverted terminal repeats (ITR) at each end (Fig. 1B)
Summary
Age-related macular degeneration (AMD) is the most common cause of blindness amongst the elderly in the industrialised world, affecting approximately 36 to 40. Supplementation of human serum with CFI was shown to dampen an over-activated complement system in high risk AMD sera by sequestering excess C3b via CFH binding (which outcompetes Factor B) and degradation of C3b required for AP C3 convertase formation [20]. It has Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice. The generation of AAV viral vector constructs carrying the cDNA of human CFI is described and successful in vitro and in vivo expression of active protein in human cell lines and murine retina is shown These encouraging pre-clinical studies have led to a first-in-human AAV gene therapy trial for late-stage AMD (NCT03846193)
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