Determination of optimal antisense oligonucleotide chemistry for efficient and safe splicing modulation in T cells

项目摘要 多发性硬化症（MS）是成年早期最常见的神经系统疾病， 导致中枢神经系统脱髓鞘和神经元损伤的自身免疫机制， 导致进行性神经功能障碍。没有治愈这种疾病的方法，目前的治疗重点 预防未来的免疫攻击，主要是通过抑制免疫系统。这导致不利的 副作用往往是严重的或致命的。因此，显然需要开发 有效和耐受性良好的治疗，以阻止MS的发展。这是一个挑战，因为MS 许多病因学和这些病因学的分子机制还不清楚。我们 揭示了MS病因的分子基础，并希望这些知识将转化为 这种特定的病因学是由于可溶性白细胞介素-α的上调引起的。 7受体（sIL 7R），这已被证明会加重疾病的进展和严重程度，在 实验性自身免疫性脑脊髓炎（EAE）小鼠MS模型，并对升高患者 几种自身免疫性疾病，包括MS、I型糖尿病、风湿性关节炎和系统性狼疮 红斑考虑到sIL-7R是由IL-7R前体mRNA中外显子6的异常排除产生的，我们 开发了一种新的生物药物，剪接调节反义寡核苷酸（SM-ASO; IL7R-005）， 纠正这种异常剪接并恢复IL 7 R蛋白同种型的正常表达。IL7R-005代表 与目前的MS疗法相比，其主要改进在于通过纠正IL 7R剪接， 致病性sIL 7R亚型，而不影响膜结合IL 7 R（mIL 7 R）的功能， 对适当的免疫功能至关重要，从而避免了当前药物的不良免疫抑制作用。不 细胞是人类中sIL 7R的主要生产者，因此为了降低sIL 7R水平，需要将IL 7R-005 在体内传递到T细胞中。尽管SM-ASO已被证明可以调节RNA剪接决定，但它可能与SM-ASO的作用有关。 在体内许多组织（例如FDA批准的Spinraza）中，SM-ASO在T细胞中的递送和功能性具有 尚未进行彻底检查，这是扩大SM-ASO治疗用途的主要障碍 免疫紊乱和新型免疫疗法的发展。在这里，我们通过以下方式解决这一障碍： 进行深入的，并排分析不同的化学修饰对 SM-ASO在原代T细胞中的效率及其在相关细胞模型中的潜在毒性作用。这一点至关重要 因为SM-ASO的化学修饰可能影响其药理学性质（例如，蜂窝 在细胞类型之间的差异性摄取），并因此定义了SM-ASO在细胞类型特异性物质中的效力。 SM-ASO的化学修饰也决定了潜在的有害作用，例如肝或肾损害。 毒性因此，这种深入的分析将能够选择具有最佳治疗效果的化学物质。 索引（即，高效力、低毒性）用于T细胞中的有效剪接调节。

PROJECT SUMMARY Multiple Sclerosis (MS) is the most common neurological disease of early adulthood and is mediated by autoimmune mechanisms that lead to demyelination and neuronal damage in the central nervous system, resulting in progressive neurological dysfunction. There is no cure for the disease and current treatments focus on preventing future immunological attacks, mainly by suppressing the immune system. This leads to adverse side effects that are often severe or fatal. Accordingly, there is a clear unmet need for the development of effective and well-tolerated therapies to arrest MS development. This has been challenging because MS has numerous etiologies and the molecular mechanisms underlying these etiologies are not well understood. We uncovered the molecular underpinnings of an MS etiology and hope this knowledge will translate into a targeted therapy for MS. This specific etiology results from up-regulation of the soluble form of the Interleukin- 7 Receptor (sIL7R), which has been shown to aggravate the progression and severity of the disease in the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS, and to be elevated in patients of several autoimmune diseases including MS, Type I diabetes, Rheumatoid arthritis and Systemic lupus erythematosus. Given that sIL7R is produced by abnormal exclusion of exon 6 from IL7R pre-mRNAs, we developed a novel biologic drug, a splicing-modulating antisense oligonucleotide (SM-ASO; IL7R-005) that corrects this abnormal splicing and restores normal expression of IL7R protein isoforms. IL7R-005 represents a major improvement over current MS therapies in that by correcting IL7R splicing, it diminishes expression of the pathogenic sIL7R isoform, without affecting the function of the membrane-bound IL7R (mIL7R), which is vital for proper immune function, thereby avoiding the adverse immunosuppressive effects of current drugs. T cells are the major producers of sIL7R in humans, and thus to reduce sIL7R levels, IL7R-005 needs to be delivered into T cells in vivo. Although SM-ASOs have been shown to modulate RNA splicing decisions in many tissues in vivo (e.g. FDA-approved Spinraza), the delivery and functionality of SM-ASOs in T cells have not been thoroughly examined, and represents the major hurdle to expand the use of SM-ASOs for treatment of immunological disorders and the development of novel immunotherapies. Here, we address this obstacle by conducting an in-depth, side-by-side analysis of the influence of diverse chemical modifications on the efficiency of SM-ASOs in primary T cells and their potential toxic effects in relevant cell models. This is critical as the chemical modifications of the SM-ASOs could influence their pharmacological properties (e.g., cellular uptake) differentially across cell-types, and thus define the potency of SM-ASOs in a cell-type specific matter. The chemical modifications of the SM-ASOs also dictate potential harmful effects, such as hepatic or renal toxicities. Therefore, this in-depth analysis will enable selection of the chemistry with the optimal therapeutic index (i.e., high potency, low toxicity) for efficient splicing modulation in T cells.