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

描述（由申请人提供）：牛卵巢超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，来维持和提高牛奶和牛肉的产量。这些胚胎可以新鲜或冷冻地移植给当地的奶牛，也可以方便地运往任何国际地点。目前的国际超排卵市场每年约为2000万美元，但由于世界范围内的饥饿、人口增长和需求，据预测，在未来十年，这一总市场可能超过2亿美元。目前的牛卵巢超排卵有许多局限性，并且恢复的活胚胎数量变化很大。到目前为止，不同批次差异的最大因素是LH激素污染和目前使用的不纯猪垂体提取物的微观异质性。美国食品药品监督管理局和其他国家监管机构禁止使用牛垂体衍生的卵泡刺激素，因为可能受到致命的牛海绵状脑病的污染，这种病在摄入后会导致人类相关的致命疾病。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，正如几十年前所有监管机构所做的那样，当时从人类垂体中提取的激素在人类中发现了类似的致命朊病毒引起的脑病。在目前的提案中，pi将：1。通过4-5个精氨酸和/或赖氨酸位点定向诱变，开发新的高亲和力重组bFSH超级激动剂类似物，以产生更有效的分子，可能延迟吸收以增加作用持续时间；对于选定的bFSH超级激动剂，还可以设计一种新的最小长度的氨基酸插入物，其中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，进一步增加作用持续时间，并产生单次注射类似物，而不会降低已增加的超级激动剂的效力/功效和降低免疫原性的潜力；最初通过瞬时转染表达这些新的类似物

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.