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

用于兽医超排卵的新型高亲和力和长效牛 FSH 类似物

• 批准号: 8450624
• 负责人: MARIUSZ W SZKUDLINSKI
• 金额: $ 15万
• 依托单位: TROPHOGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8450624
• 关键词: Novel; Affinity; Long; Acting; Bovine

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.

描述（由申请人提供）：牛卵巢超排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种鸡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。这些胚胎可以新鲜或冷冻移植到当地受体牛身上，也可以方便地运送到任何国际地点。目前超排卵的国际市场每年约为 2000 万美元，但由于全球饥饿、人口增长和需求，预计未来十年这一市场总额可能会超过 2 亿美元。当前牛卵巢超排卵存在许多局限性，并且回收的存活胚胎数量存在很大差异。到目前为止，不同批次差异的最大因素是目前使用的猪垂体不纯提取物中 LH 激素污染​​和微异质性。 FDA 和其他国家监管机构已禁止使用牛垂体衍生的 FSH，因为可能会受到致命的牛海绵状脑病的污染，而人类摄入后会导致相关的致命疾病。一些欧洲国家还禁止使用来自任何物种的任何兽用垂体产品，就像几十年前所有监管机构所做的那样，当时在人类中发现了一种类似的由人类垂体衍生的激素引起的致命性朊病毒脑病。在当前提案中，PI 将： 1. 通过最少的 4-5 精氨酸和/或赖氨酸定点诱变开发新型高亲和力重组 bFSH 超级激动剂类似物，以产生更有效的分子，并可能延迟吸收以延长作用持续时间；对于选定的 bFSH 超级激动剂，还设计了一种新型最小长度氨基酸插入物，其中包含一个或两个复合碳水化合物新糖基化位点，以延长半衰期，进一步增加作用持续时间，并产生单次注射类似物，而不会降低超级激动剂的效力/功效以及降低免疫原性的可能性；最初通过瞬时转染表达这些新颖的类似物 一种先前优化的、新颖的、高表达且低成本的生产方法。 2. 部分纯化通过瞬时转染表达的所选 4-6 个类似物，并通过先前验证的 bFSH 类似物 ELISA 免疫测定、稳健的体外生物测定、SDS-PAGE 电泳和等电聚焦凝胶分析进行体外表征。 3. 选择 3-5 个最佳 bFSH 类似物候选物，通过小鼠和/或大鼠的 pK 研究以及卵巢重量增加的经典 Steelman-Pohley FSH 生物测定以及窦卵泡数量和大小的组织学分析进行体内分析。 4. 根据体内分析，选择最佳的2-3个候选类似物构建双顺反子表达载体，并启动CHO-DG44细胞中类似物表达的扩增，为2期大规模生产、纯化和超排研究做好准备 牛。这种全新的重组 bFSH 类似物受国际专利保护，直至 2032 年，并且在美国没有正在开发的已知重组竞争产品，应以大大降低的成本提高牛超数排卵的功效、便利性和安全性。 公共卫生相关性：通过激素刺激母牛生产许多鸡蛋和胚胎，称为超排卵，是当前维持和提高牛奶和牛肉产量以应对全球饥饿和人口增长的最先进方法。目前使用的不纯刺激激素（称为 FSH，源自猪垂体）有许多局限性，包括可能受到蛋白质感染剂的污染，这种感染剂在人类食用这种受感染的肉类后会导致 100% 致命的脑部疾病。两位研究人员发明了一种全新的、更活跃的 DNA 衍生形式的类似牛的 FSH，它不含任何感染因子，并且应该以大大降低的成本提高牛超数排卵的功效、便利性和安全性。