Rapid egg bioreactor construction for Fabry disease therapeutics

摘要 法布里病是一种溶酶体贮积病（LSD），影响1/40，000至117，000活产。的 目前的治疗是用重组人α-半乳糖苷酶A（GalA）的酶替代疗法（ERT）。一 这种治疗的主要医疗负担是每患者年35万美元的高药物成本。 为了降低法布里病的药物成本，我们建议开发一种快速构建成本的方法- 用于GalA生产的有效鸡蛋生物反应器，其具有生产50 mg重组体的潜力 每个鸡蛋的蛋白质不到10美元。此前，曾为另一种LSD开发过类似的卵子生物反应器， 溶酶体酸性脂肪酶（LAL）缺乏。用于LAL缺乏症的药物sebelipase alfa一直是FDA唯一的- 从转基因鸡蛋中提取的药物。 传统的鸡胚盘转基因技术因其效率低、鸡只数以千计而受到限制 将被筛选以获得阳性克隆。原始生殖细胞（PGCs）的另一种主流方法 患有PGCs对生殖系而不是体细胞有贡献的事实，并且仅产生雄性G 0 嵌合体，延迟评估嵌合体，直到六个月后，当他们的精液基因分型。我们 一种新的方法将产生具有体细胞和生殖系嵌合的嵌合公鸡和母鸡， 大大加快了转基因鸡的建设。 在第一阶段，我们将用GalA表达盒基因敲入鸡细胞系， 转录OVA基因座，构建嵌合体鸡。嵌合体母鸡将为快速繁殖提供鸡蛋。 GalA丰度和活性的评估。这种验证将表明这种方法的可行性 具有杂合和纯合OVA-GalA基因敲入的鸡的后代。

Abstract Fabry 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.