Targeted Enzyme Replacement Therapy for Rare Forms of Osteogenesis Imperfecta

描述（由申请人提供）：本项目的长期目标是开发一种有效的蛋白质替代治疗（PRT），用于患有罕见形式的成骨肥大、OI-VIII（脯氨酰3-羟化酶1缺乏症; P3 H1）和OI-VII（软骨相关蛋白CRTAP缺乏症）的患者。这些区域重建技术将把植物生物生产的安全性和成本优势与加强交付和疾病矫正的创新结合起来。骨质疏松症是一组遗传性结缔组织疾病，其特征是低骨量和骨脆性增加。目前OI的治疗选择仅限于双膦酸盐治疗，其抑制骨吸收，但由于对年轻患者以及成人的有害影响的可能性而存在争议。为这些患者开发有效的PRT具有挑战性，因为经典的酶替代疗法（Erts）在纠正骨相关病变方面相对无效。这些罕见OI疾病的PRT将 需要有效递送至多个间充质来源的细胞谱系以及有效靶向粗面内质网（rER）以纠正胶原畸形。为了扩大校正蛋白质向关键细胞和组织的递送，BioStrategies LC一直在测试 几种植物凝集素作为PRT's进入疾病细胞的有效载体的潜力。这些凝集素对哺乳动物细胞表面常见的糖蛋白和糖脂具有高亲和力，并介导有效的细胞摄取、胞吞转运和递送至靶组织。我们已经证明，这些凝集素之一，RTB，有效地进行大融合模型蛋白到多个细胞的骨骼系统，包括软骨细胞，成骨细胞和间充质细胞。我们还表明，RTB，修改包含一个KDEL ER检索信号（RTBER），有效地指导负载蛋白质的ER后的内吞作用。我们假设我们的融合蛋白将进入骨骼系统的细胞，并将递送PRT以纠正OI-VIII和OI-VII中的胶原蛋白缺陷。该SBIR I期可行性研究的目标是测试植物产生RTBER融合物与P3 H1和/或CRTAP的潜力，所述融合物被吸收到成骨细胞中，动员到rER，并在细胞水平上发挥纠正胶原蛋白加工缺陷的功能。I期的具体目标是1）使用基于植物的瞬时表达平台生产和表征RTB：胶原修饰蛋白融合体，2）通过OI细胞中的疾病表型校正在体外证明产品功效。基于在罕见隐性OI的OI-VIII或OI-VI模型中成功满足体外细胞校正的I期里程碑，后续II期研究将集中于这些OI疾病的小鼠模型中的体内功效。这些研究将包括在P3 H1缺陷或Crtap-/-小鼠中进行长期替代治疗后的短期生物分布分析和骨骼表型评价，导致IIB期毒理学评估和其他临床前终点，以支持FDA的IND申请。

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop an effective protein replacement therapeutic (PRT) for patients affected with rare forms of Osteogenesis imperfecta, OI-VIII (deficiency in prolyl 3-hydroxylase 1; P3H1) and OI-VII (deficiency in cartilage associated protein CRTAP). These PRTs will integrate the safety and cost advantages of plant-based bioproduction with innovations that enhance delivery and disease correction. OI is a group of genetic connective tissue disorders characterized by low bone mass and increased bone fragility. Current treatment options for OI are limited to bisphosphonates treatments, which inhibit bone resorption but are controversial due to the likelihood of deleterious effects in young patients as well as adults. Developing effective PRTs for these patients is challenging since classic enzyme replacement therapies (ERTs) have been relatively ineffective in correcting bone-related pathologies. PRTs for these rare OI diseases will require efficient delivery to multiple mesenchyme-derived cell lineages as well as effective targeting to rough endoplasmic reticulum (rER) to correct collagen malformation. To expand the delivery of corrective protein to critical cells and tissues, BioStrategies LC has been testing the potential of several plant lectins to function as effective carriers of PRT's into disease cells. These lectins have high affinity for glycoproteins and glycolipids common on mammalian cell surfaces and mediate efficient cellular uptake, transcytosis, and delivery to target tissues. We have demonstrated that one of these lectins, RTB, efficiently carries large fused model proteins into multiple cells of the skeletal systems including chondrocytes, osteoblasts, and mesenchymal cells. We also showed that RTB, modified to contain a KDEL ER-retrieval signal (RTBER), effectively directs payload proteins to the ER following endocytosis. We hypothesize that our fusion proteins will access cells of the skeletal system and will deliver PRTs to correct collagen defects in OI-VIII and OI-VII. The goal of this SBIR Phase I feasibility study is to test the potential of plants to produce RTBER fusions with P3H1 and/or CRTAP that are taken up into osteogenic cells, mobilized to the rER, and function to correct collagen processing defects at the cellular level. Specific aims for Phase I are 1) to produce and characterize RTB:collagen-modifying protein fusions using a transient plant-based expression platform, and 2) to demonstrate product efficacy in vitro by disease phenotype correction in OI cells. Based on successfully meeting the Phase I milestones of in vitro cell correction in either OI-VIII or OI- VI models of rare recessive OI, follow-on Phase II studies would focus on in vivo efficacy in mouse models of these OI diseases. These studies would include short-term biodistribution analyses and skeletal phenotype evaluation following extended replacement therapy treatments in either P3H1 deficient or Crtap-/- mice leading to Phase IIB toxicology assessment and other preclinical endpoints in support of an IND application at FDA.