Novel High Affinity and Long-Acting Bovine FSH Analogs for Veterinary Superovulat

描述(申请人提供)：牛卵巢超数排卵是目前世界范围内采用的最先进的方法，通过遗传选择最优质的育种者和最优质的胚胎进行移植，以维持和提高牛奶和牛肉的产量。这些胚胎既可以是新鲜的，也可以是冷冻的，既可以转移到当地的受体奶牛，也可以方便地运往任何国际地点。目前国际超促排卵市场每年约2000万美元，但由于全球饥饿、人口增长和需求，预计未来十年这一总市场可能超过2亿美元。目前的牛卵巢超数排卵存在许多局限性，回收的可用胚胎数量也存在很大差异。到目前为止，造成不同批次差异的最大因素是促黄体激素污染和目前使用的猪脑垂体不纯提取物中的微观异质性。FDA和其他国家监管机构已禁止使用牛脑垂体源性FSH，因为可能会受到致命性牛海绵状脑病的污染，牛海绵状脑病在摄入后会导致一种相关的致命疾病。几个欧洲国家还禁止使用来自任何物种的任何兽用脑垂体产品，就像几十年前所有监管机构发生的那样，当时在人类身上发现了一种类似的致命的普里恩引起的脑病，这种疾病是由人类脑下垂体中提取的激素引起的。在目前的建议中，PI将：1.通过最小4-5精氨酸和/或赖氨酸定点突变来开发新型高亲和力重组bFSH超激动剂类似物，以产生更强大和有效的分子，并可能延迟吸收以增加作用持续时间；对于选定的bFSH超激动剂，还设计了一种新的最小长度的氨基酸插入，包含一个或两个复杂的碳水化合物新糖基化位点，以延长半衰期，进一步延长作用时间，并产生一次注射类似物，而不降低增加的超激动剂效力/效力和降低的免疫原性；最初通过瞬时转染表达这些新的类似物 一种经过优化的、新颖的、高表达和低成本的生产方法。2.通过瞬时转染法对筛选出的4-6类似物进行部分纯化，并通过前期验证的bFSH类似物的ELISA法、Robust体外生物法、SDS-PAGE电泳法和等电聚焦凝胶电泳法对其进行体外鉴定。3.通过小鼠和/或大鼠的PK研究，以及经典的Steelman-Pohley FSH体重增加生物测定法和有腔卵泡数量和大小的组织学分析，选择3-5个最佳的bFSH类似物进行活体分析。4.在体内分析的基础上，选择最佳的2-3个候选类似物构建双顺反子表达载体，并在CHO-DG44细胞中启动类似物表达的扩增，为第二阶段大规模生产、纯化和超排研究做准备。 牛群。这种全新的重组bFSH类似物受国际专利保护，直到2032年&在美国没有已知的重组竞争产品正在开发中，应该会以大大降低的成本提高牛超排的效率、便利性和安全性。 公共卫生相关性：通过荷尔蒙刺激在母牛身上生产许多鸡蛋和胚胎，称为超数排卵，是目前最先进的方法，可以保持和提高牛奶和牛肉的产量，以应对全球饥饿和人口增长。目前使用的被称为FSH的不纯刺激素存在许多限制，包括可能被一种蛋白质感染剂污染，这种病毒在食用受感染的肉类后会导致100%致命的人类脑部疾病。这两位研究人员已经发明了一种全新的、更具活性的类似奶牛的FSH的DNA衍生形式，它不含任何感染剂，应该会在大大降低成本的情况下提高奶牛超数排卵的效率、便利性和安全性。

DESCRIPTION (provided by applicant): Bovine ovarian superovulation is the current state-of-the-art method employed worldwide to maintain and improve milk and beef production through genetic selection of the best quality breeders and best quality embryos for transfer. These embryos may either be transferred fresh or frozen both to local recipient cows as well as conveniently shipped to any international locale. The current international market for superovulation is about $20 million per year but because of worldwide hunger, population growth and demand it has been predicted that this total market may exceed $200 million in the next decade. There are many limitations of current bovine ovarian superovulation and wide variations of the number of viable embryos recovered. By far the greatest factors in the variability of different lots are LH hormone contamination and microheterogeneity in currently used impure extracts of porcine pituitaries. Use of bovine pituitary-derived FSH has been banned by FDA and other national regulatory agencies because of possible contamination by fatal bovine spongiform encephalopathy which causes a related fatal disease in humans after ingestion. Several European countries have additionally banned the use of any veterinary pituitary products from any species, as had occurred decades earlier by all regulatory agencies when a similar fatal prion-caused encephalopathy had been discovered in humans from hormones derived from human pituitaries. In the current proposal the PIs will: 1. Develop novel high affinity recombinant bFSH superagonist analogs by minimal 4-5 arginine and/or lysine site-directed mutagenesis to produce a more potent and efficacious molecule with possible delayed absorption to increase duration of action; for selected bFSH superagonists also engineer a novel minimal length amino acid insert containing one or two complex carbohydrate neoglycosylation sites to increase half life to further increase duration of action and produce a single injection analog without reducing increased superagonist potency/efficacy plus reduced potential for immunogenicity; initially express these novel analogs by transient transfection using a previously optimized, novel, high expression & low cost production method. 2. Partially purify selected 4-6 analogs expressed by transient transfection and characterize in vitro by previously validated bFSH analog ELISA immunoassay, robust in vitro bioassay, SDS-PAGE electrophoresis and isoelectric focusing gel analysis. 3. Select the 3-5 best bFSH analog candidates to analyze in vivo by pK studies in mice and/or rats as well as the classic Steelman-Pohley FSH bioassay of ovarian weight gain plus histological analysis of antral follicle number and size. 4. Based on in vivo analysis, select the best 2-3 candidate analogs to construct dicistronic expression vectors, and initiate amplification of analogs expression in CHO-DG44 cells in preparation for Phase 2 large-scale production, purification and superovulation studies in cattle. This completely novel recombinant bFSH analog protected by international patents until 2032 & without a known recombinant competing product under development in the US should increase the efficacy, convenience & safety of bovine superovulation at much reduced cost. PUBLIC HEALTH RELEVANCE: Production of many eggs and embryos by hormone stimulation in female cows, termed superovulation, is the current state-of-the-art method to maintain and improve milk and beef production in response to worldwide hunger and population growth. There are many limitations of the current impure stimulating hormone used, called FSH, derived from pig pituitaries, including possible contamination by a protein infectious agent which causes a 100% fatal brain disease in humans after eating such infected meat. The two investigators have invented a completely new and more active DNA - derived form of cow-like FSH which cannot contain any infectious agent & should increase the efficacy, convenience & safety of cow superovulation at much reduced cost.