Chimeric TGF-beta Ligands for Regenerative Medicine

 描述(由申请人提供)：再生医学为治疗多种疾病提供了巨大的希望，在控制和指导细胞分化以组织修复为目的的能力方面取得了重大进展。然而，发育信号机制是复杂的，体外细胞分化和体内组织再生的方案还需要改进。转化生长因子超家族成员包括激活素、结节、生长分化因子和骨形态发生蛋白，它们在发育和组织形态发生中起着重要作用，在再生医学中得到了广泛的应用。本研究的目的是对转化生长因子超家族配体进行结构导向的修饰，以开发新的信号活性，增强有效的再生药物应用于肌肉骨骼疾病所需的细胞分化步骤。在自然脊椎动物的发育过程中，中胚层的规范是由Nodal信号驱动的，代表着多能前体细胞向骨、肌腱和软骨等多种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，由于它们完全忽略了Nodal信号，因此很可能会得到显着改进。这项提议的目标1将产生具有一系列信号活性的嵌合Nodal和Activin分子，然后我们将使用它们来微调体外中胚层分化。Nodal和Activin复合体与其I型(ALK4)和II型(ActRII和ActRIIB)信号受体以及共同受体Cripto的组装的结构和机械基础也将被确定，所产生的信息将被用于帮助设计嵌合配体。骨细胞、软骨细胞和腱细胞的定向终末分化分别是骨、软骨和肌腱再生的关键。目前用于确定这些细胞命运的协议使用高剂量的特定BMP和GDF。目的2验证以下假设：通过用低亲和力的II型受体结合表位取代激活素A的高亲和力结合表位来提高BMPs/GDF的效力，将产生具有更强的促进骨、软骨和腱细胞终末分化能力的工程配体。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型受体的结合，使这些配体对此目的更加有效。总体而言，这些研究不仅为发现治疗肌肉骨骼疾病的潜在有效疗法铺平了道路，而且还将阐明转化生长因子超家族配体信号传递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.