Chimeric TGF-beta Ligands for Regenerative Medicine

 描述（由申请人提供）：再生医学在治疗各种疾病方面具有很大的前景，并且在控制和指导细胞分化以实现组织修复的能力方面取得了重大进展。然而，发育信号传导机制是复杂的，体外细胞分化方案和体内组织再生需要大量改进。TGF-β超家族的成员，包括激活素、结节、生长和分化因子（GDFs）和骨形态发生蛋白（BMP），在发育和组织形态发生过程中起关键作用，并已广泛用于再生医学应用。本研究的目的是对TGF-β超家族配体进行结构导向的修饰，以开发新的信号传导活性，增强肌肉骨骼疾病中有效再生药物应用所需的细胞分化步骤。中胚层特化在自然脊椎动物发育期间由Nodal信号传导驱动，并且代表多能祖细胞向包括骨、肌腱和软骨的许多谱系分化的关键早期步骤。目前用于体外中胚层分化的方案使用不同浓度的激活素A，并且可能会显著改善，因为它们完全忽略了Nodal信号传导。本提案的目的1将产生具有一系列信号传导活性的嵌合Nodal和激活素分子，然后我们将使用它们来微调体外中胚层分化。还将确定Nodal和激活素复合物与其I型（ALK 4）和II型（ActRII和ActRIIB）信号传导受体和共受体Cripto组装的结构和机制基础，并且所得信息将用于辅助工程化嵌合配体。骨细胞、软骨细胞和肌腱细胞的定向终末分化分别是骨、软骨和肌腱再生的关键。目前用于指定这些细胞命运的方案使用高剂量的特异性BMP和GDF。目的2将检验如下假设：通过用BMP/GDF的低亲和力II型受体结合表位取代活化素A的高亲和力结合表位来增强BMP/GDF的效力，将产生具有增强的促进骨细胞、软骨细胞和腱细胞的终末分化的能力的工程化配体。它还将测试额外的结构导向修饰是否可以进一步增加I型或II型受体结合，使这些配体更有效地用于此目的。总的来说，这些研究不仅为发现肌肉骨骼疾病的潜在强大治疗方法铺平了道路，而且还阐明了TGF-β超家族配体信号传导的关键结构和机制方面。

 DESCRIPTION (provided by applicant): Regenerative medicine holds great promise for the treatment of a wide range of diseases and major advances have been made in the ability to control and direct cellular differentiation for the purpose of tissue repair. However, the developmental signaling mechanisms are complex and cellular differentiation protocols in vitro and tissue regeneration in vivo require much improvement. Members of the TGF-ß superfamily including Activins, Nodal, Growth and Differentiation Factors (GDFs) and Bone Morphogenetic Proteins (BMPs) play crucial roles during development and tissue morphogenesis and have been widely used in regenerative medicine applications. The goal of the present study is to make structure-guided modifications of TGF-ß superfamily ligands in order to develop new signaling activities that enhance cellular differentiation steps required for effective regenerativ medicine applications in musculoskeletal disorders. Mesodermal specification is driven by Nodal signaling during natural vertebrate development and represents a critical early step in the differentiation of pluripotent progenitor cells toward many lineages including bone, tendon and cartilage. Current protocols for in vitro mesodermal differentiation use varying concentrations of Activin A and can likely be improved significantly as they neglect Nodal signaling altogether. Aim 1 of this proposal will generate chimeric Nodal and Activin molecules with a range of signaling activities that we will then use to fine tune mesodermal differentiation in vitro. The structural and mechanistic basis of Nodal and Activin complex assembly with their type I (ALK4) and type II (ActRII and ActRIIB) signaling receptors and the co-receptor Cripto will also be determined and the resulting information will be used to assist in engineering the chimeric ligands. Directed terminal differentiation of osteocytes, chondrocytes and tenocytes is critical fo regeneration of bone, cartilage and tendon, respectively. Current protocols for specifying these cell fates use high doses of specific BMPs and GDFs. Aim 2 will test the hypothesis that enhancing the potency of BMPs/GDFs by substituting their low affinity type II receptor binding epitopes for the high affinity binding epitope of Activin A will result in engineered ligands with heightened ability to promote terminal differentiation of osteo-, chondro- and tenocytes. It will also test if additional structure-guided modifications can further increase type I or type II receptor binding to make these ligands even more effective for this purpose. Overall, these studies will not only pave the way towards discovering potentially powerful therapeutics for musculoskeletal diseases but also elucidate key structural and mechanistic aspects of signaling of TGF-ß superfamily ligands.