Rapid egg bioreactor construction for Fabry disease therapeutics

AbstractFabry disease is a lysosomal storage disease (LSD) affecting 1 in 40,000 to 117,000 live births. The current treatment is enzyme replacement therapy (ERT) with recombinant human α-galactosidase A (GalA). A major healthcare burden from this treatment is the high drug cost at $350k per patient-year.To reduce the drug cost for Fabry disease, we propose to develop a rapid approach to construct cost- effective chicken egg bioreactor for GalA production, which has the potential to produce 50 mg recombinant protein per egg for less than $10. Previously, a similar egg bioreactor was developed for another LSD, lysosomal acid lipase (LAL) deficiency. The drug sebelipase alfa for LAL deficiency has been the only FDA- approved therapeutics from transgenic chicken eggs.The traditional blastoderm transgenesis of chicken is limited by its efficiency and thousands of chickens will be screened to obtain a positive clone. Another mainstream approach with primordial germ cells (PGCs) suffers from the fact that PGCs contribute to the germline but not somatic cells, and only generate male G0 chimeras, delaying the assessment of chimerism until six months later when their semen is genotyped. Our novel approach will generate chimeric roosters and hens with both somatic and germline chimerism, which greatly accelerate transgenic chicken construction.In Phase I, we will engineer chicken cell lines with GalA expression cassette knock-in at the highly transcribed OVA locus and construct the chimeric chickens. The chimeric hens will provide eggs for rapid assessment of GalA abundance and activity. This validation will show the feasibility of this approach for the future generation of chicken with heterozygous and homozygous OVA-GalA knock-in.Project NarrativeIn the present work, we propose to develop a chicken egg bioreactor producing human α-galactosidase A, an essential enzyme to treat Fabry disease, with our novel rapid transgenic chicken construction approach. The success of the project will significantly reduce the production cost of this therapeutic enzyme and increase its supply security and availability and therefore is of great commercial value. The proposed transgenic chickens also have the potential to produce a wide range of other highly valuable protein therapeutics to lower the burden of the US healthcare system.