Targeted Enzyme Replacement Therapy for Rare Forms of Osteogenesis Imperfecta

描述(由申请人提供)：该项目的长期目标是开发一种有效的蛋白质替代疗法(PRT)，用于治疗罕见形式的成骨不全、OI-VIII(Prolyl 3-羟基酶1缺乏；P3H1)和OI-VII(软骨相关蛋白CRTAP缺乏)。这些PRT将把以植物为基础的生物生产的安全和成本优势与加强交付和疾病纠正的创新结合起来。OI是一组遗传性结缔组织疾病，以骨量减少和骨脆性增加为特征。目前OI的治疗选择仅限于抑制骨吸收的双膦酸盐治疗，但由于可能对年轻患者和成年人产生有害影响，因此存在争议。为这些患者开发有效的PRT是具有挑战性的，因为经典的酶替代疗法(ERT)在纠正与骨相关的病理方面相对无效。这些罕见的OI疾病的PRT将 需要高效地输送到多个间充质来源的细胞系，以及有效地靶向粗面内质网(RER)以纠正胶原畸形。为了将纠正蛋白的输送扩大到关键细胞和组织，BioStrategy LC一直在测试 几种植物凝集素作为PRT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很高的亲和力，并介导有效的细胞摄取、跨细胞和递送到靶组织。我们已经证明，其中一种凝集素，RTB，有效地将大的融合模型蛋白携带到骨骼系统的多个细胞中，包括软骨细胞、成骨细胞和间充质细胞。我们还发现，RTB被修饰为包含KDEL ER检索信号(RTBER)，在内吞作用后有效地将有效载荷蛋白质定向到ER。我们假设我们的融合蛋白将进入骨骼系统的细胞，并将传递PRT来纠正OI-VIII和OI-VII中的胶原缺陷。这项SBIR第一阶段可行性研究的目标是测试植物与P3H1和/或CRTAP产生RTBER融合的潜力，这些融合被吸收成成骨细胞，动员到RER，并在细胞水平上纠正胶原加工缺陷。第一阶段的具体目标是1)生产和表征RTB：使用基于植物的瞬时表达平台的胶原修饰蛋白融合，以及2)通过在OI细胞中纠正疾病表型来证明体外产品的有效性。在成功达到OI-或OI-VI罕见隐性OI模型体外细胞矫正的第一阶段里程碑的基础上，后续的第二阶段研究将专注于这些OI疾病的小鼠模型的体内疗效。这些研究将包括P3H1缺陷小鼠或CrTAP-/-小鼠在延长替代疗法治疗后的短期生物分布分析和骨骼表型评估，从而进行IIB期毒理学评估和其他临床前终点，以支持FDA的IND应用。

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.