Development of Superactive Analogs of LH and FSH

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DESCRIPTION (provided by applicant): Our previous structure-function studies resulted in the development of the first analogs of glycoprotein hormones with major increases in receptor binding affinity and bioactivity ("super active analogs"). More recently, we have further optimized human luteinizing hormone (LH) and human follicle-stimulating hormone (FSH) analogs. However, in contrast to human thyroid-stimulating hormone (TSH) analogs, we have not yet studied bioactivity of these analogs in vivo. Such animal studies with therapeutically relevant end-points are critical to demonstrate potential therapeutic utility of newly developed LH and FSH analogs. Since the gonadotropin in vivo bioassays require higher quantities of hormones than that obtainable from transient transfection experiments we propose in Aim 1 to develop cell lines producing high levels of candidate super active analogs and produce quantities sufficient for initial animal studies. This Aim will consist of construction of dicistronic vectors with amplifiable markers and mutated subunit cDNAs, amplification process, production and concentration of unpurified analogs. In Aim 2 we propose to perform initial experiments using currently established rodent in vivo bioassays modified to study the effect of LH and FSH on oocyte production, ovarian weight and steroidogenesis. The Aim 2 will also include respective LH and FSH bioassays with four selected analogs, two unmodified hormones and two mock controls. The results of such in vivo bioassays will be essential in the final selection of candidate analogs for high-level production and purification, as well as subsequent phase 2 primate and toxicology studies.