Rapid egg bioreactor construction for Fabry disease therapeutics

摘要 法布里病是一种溶酶体储积症(LSD)，每40,000至117,000名活产儿中就有一名受到影响。这个 目前的治疗方法是用重组人α-半乳糖苷酶A(GALA)进行酶替代治疗。一个 这种治疗带来的主要医疗负担是高昂的药品成本，每名患者每年35万美元。 为了降低治疗法布里病的药物成本，我们建议开发一种快速方法来构建成本- 生产GALA的高效鸡蛋生物反应器，有可能生产50 mg重组 每个鸡蛋的蛋白质价格不到10美元。此前，另一种迷幻药也开发了类似的鸡蛋生物反应器， 溶酶体酸性脂肪酶(LAL)缺乏症。治疗LAL缺乏症的药物Sebelipase Alfa一直是唯一的FDA- 转基因鸡蛋经批准的治疗方法。 传统的鸡胚层转基因技术受到效率和成百上千只鸡的限制。 将进行筛选以获得阳性克隆。另一种主流方法是原始生殖细胞(PGCs) 受累于PGCs对生殖系的贡献，而不是体细胞，并且只产生雄性G0 嵌合体，将嵌合体的评估推迟到六个月后，当他们的精子进行基因分型时。我们的 新的方法将产生具有体细胞和生殖系嵌合体的嵌合公鸡和母鸡，这 大大加快了转基因鸡的构建。 在第一阶段，我们将用gala表达盒敲入高度 转录OVA基因座，构建嵌合鸡。嵌合母鸡将为快速提供鸡蛋 评估联欢晚会的丰富性和活跃性。这一验证将表明该方法对于 具有杂合子和纯合子OVA-GALA敲入的后代鸡。

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.