Chemically Modified Peptide Agents for Next-Generation Conjugate Therapies to Treat Duchenne Muscular Dystrophy

项目摘要 杜氏肌营养不良症(DMD)是一种严重的疾病，大约每3500名男孩中就有一人受到影响，导致 随着时间的推移，严重的肌肉无力和退化。平均预期寿命为26岁， 死亡通常是由心肌病或呼吸道感染造成的。治疗选择非常有限 是为了治疗这些儿童而存在的。DMD是由Dstrophin编码基因的突变引起的 蛋白。一种有希望的治疗方法是跳过外显子，这是一种反义 寡核苷酸诱导包含突变的外显子从最终的dystrophin mRNA转录本中剪接出来。 FDA最近暂时批准了第一种也是唯一一种针对DMD的治疗方法，Eteplirsen，它可以进行 它通过外显子跳跃的方式发挥作用。Eteplirsen属于一类反义治疗药物，称为 磷酸二酯吗啉寡核苷酸(PMO)。尽管PMO是诱人的触发分子 外显子跳过和dystrophin修复，它们的临床疗效受到细胞间传递能力差的限制 并进入到细胞核中。多肽在促进货物的核运输方面显示出了希望。 然而，目前只探索了有限数量的多肽序列来传递PMO和 多肽在血清中容易被蛋白质降解。因此，我建议开发一种基于多肽的递送方式 产生新的PMO递送代理的平台，将增强核递送和外显子跳过 提高血清中的稳定性。一种方法是创建线性和分支的多肽嵌合体 序列，以探索多肽序列和结构多样性如何改善PMO的传递。 同时，第二种方法将是创建具有富氟连接物的大环肽，如 大周期通常在稳定性和交付方面带来好处。将对这两种方法进行评估 无论是在报告PMO的细胞绿色荧光蛋白分析中，还是在来自 用治疗性PMO建立DMD小鼠模型。在这项工作中产生的化合物将成为下一步的线索- 新一代共轭DMD疗法，提高了临床疗效。更广泛地说，考虑到 将这些基于多肽的支架连接到其他感兴趣的货物上，我设想这项工作可以 易于应用于改善先天性疾病的其他治疗方法，其中主要限制因素是 细胞内传递。

Project Summary Duchenne muscular dystrophy (DMD) is a severe disease affecting approximately 1 in 3500 boys, causing profound muscle weakness and degeneration over time. The average life expectancy is 26 years of age and death is typically a result of either cardiomyopathy or respiratory infection. Very limited therapeutic options exist for the treatment of these children. DMD is caused by mutations in the gene encoding the dystrophin protein. One promising approach to treatment involves exon skipping, a process in which an antisense oligonucleotide induces the mutation-containing exon to be spliced out of the final dystrophin mRNA transcript. The FDA recently provisionally approved the first and only DMD-specific therapy, Eteplirsen, which carries out its effect via exon skipping. Eteplirsen belongs to a class of antisense therapeutics known as phosphorodiamidate morpholino oligonucleotide (PMO). Although PMOs are attractive molecules to trigger exon skipping and dystrophin restoration, their clinical efficacy has been limited by poor delivery across the cell membrane and into the nucleus. Peptides have shown promise in facilitating the nuclear delivery of cargoes. However, only a limited number of peptide sequences have been explored for the delivery of PMOs and peptides are prone to proteolytic degradation in serum. Thus, I propose to develop a peptide-based delivery platform to generate new agents for the delivery of PMOs that will enhance nuclear delivery and exon skipping and improve stability in serum. One approach will be to create linear and branched chimeras of peptide sequences, in order to explore how peptide sequence and structural diversity can improve PMO delivery. Simultaneously, a second approach will be to create macrocyclic peptides with fluorine-rich linkers, as macrocycles often confer benefits in terms of stability and delivery. These two approaches will be evaluated both in a cellular green fluorescent protein assay with a reporter PMO and in skeletal muscle cells from a mouse model of DMD with a therapeutic PMO. The compounds generated in this work will be leads for next- generation conjugate DMD therapies with improved clinical efficacy. More generally, given the ease of conjugation of these peptide-based scaffolds to other cargoes of interest, I envision that this work can be readily applied to improve other therapies for congenital diseases in which the major limiting factor is intracellular delivery.