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

确定最佳反义寡核苷酸化学，以实现 T 细胞中高效、安全的剪接调节

• 批准号: 9907140
• 负责人: Gaddiel Galarza-Munoz
• 金额: $ 28.37万
• 依托单位: AUTOIMMUNITY BIOLOGIC SOLUTIONS, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-05 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907140
• 关键词: Address; Affect; Animal Model; Antisense Oligonucleotides; Autoimmune Diseases; Autoimmune Process; Award; Biological; Biological Assay; Callithrix; Cell model; Cells; Chemicals; Chemistry; Clinic; Clinical Research; Cognitive; Complex; Cultured Cells; Demyelinations; Development; Diagnosis; Disease; Emotional; Etiology; Exclusion; Exhibits; Exons; Experimental Autoimmune Encephalomyelitis; FDA approved; Fruit; Funding; Future; Generations; Genetic; Genetic Diseases; Goals; Grant; Hepatic; Hepatotoxicity; Human; IL7R gene; Immune; Immune System Diseases; Immune system; Immunologics; Immunomodulators; Immunosuppression; Immunotherapy; Insulin-Dependent Diabetes Mellitus; Interleukin 7 Receptor; Investigation; Kidney; Knowledge; Lead; Measures; Mediating; Medical; Membrane; Modeling; Modification; Molecular; Multiple Sclerosis; Neuraxis; Neurologic Dysfunctions; Neurons; Pathogenicity; Patients; Pharmaceutical Preparations; Pharmacology; Phase; Property; Protein Isoforms; RNA Splicing; Research; Rheumatoid Arthritis; Safety; Sensory; Severity of illness; Side; Small Business Innovation Research Grant; Small Business Technology Transfer Research; Systemic Lupus Erythematosus; T-Lymphocyte; Testing; Therapeutic; Therapeutic Index; Therapeutic Intervention; Tissues; Toxic effect; Toxicology; Translating; Up-Regulation; Work; base; cell type; emerging adult; immune function; improved; in vivo; in vivo evaluation; mRNA Precursor; motor impairment; mouse model; nephrotoxicity; nervous system disorder; neuron loss; novel; personalized medicine; preclinical study; prevent; side effect; success; targeted treatment; tool; uptake

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.

项目总结 多发性硬化症(MS)是最常见的成年早期神经系统疾病，由 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。这种疾病没有治愈的方法，目前的治疗重点是 关于预防未来的免疫攻击，主要是通过抑制免疫系统。这导致了不利的 通常是严重或致命的副作用。因此，有一种明显未得到满足的发展需要。 有效和耐受性良好的治疗方法阻止多发性硬化症的发展。这很有挑战性，因为微软已经 许多病因和这些病因背后的分子机制还不是很清楚。我们 揭示了多发性硬化症病因学的分子基础，并希望这一知识将转化为 多发性硬化症的靶向治疗这种特殊的病因是由于白介素2的可溶性形式上调所致。 7受体(SIL7R)，这已被证明是加剧疾病的进展和严重程度在 实验性自身免疫性脑脊髓炎(EAE)小鼠模型MS，并将在患者中升高 几种自身免疫性疾病，包括多发性硬化症、I型糖尿病、类风湿关节炎和系统性狼疮 红斑狼疮。鉴于sIL7R是通过IL7R前mRNAs中外显子6的异常排除而产生的，我们 开发了一种新的生物药物，一种剪接调节的反义寡核苷酸(SM-ASO；IL7R-005)， 纠正这种异常剪接，恢复IL7R蛋白亚型的正常表达。IL7R-005代表 相对于目前的多发性硬化症治疗方法的一个重大改进是，通过纠正IL7R剪接，它减少了 致病的sIL7R亚型，而不影响膜结合的IL7R(MIL7R)的功能，即 对正常的免疫功能至关重要，从而避免现有药物的不良免疫抑制作用。T 细胞是人类sIL7R的主要产生者，因此为了降低sIL7R水平，IL7R-005需要 在体内导入T细胞。尽管SM-ASO已被证明可以调节RNA剪接决定 体内许多组织(如FDA批准的Spinraza)，SM-ASOS在T细胞中的传递和功能 没有得到彻底的检查，是扩大SM-ASOS用于治疗的主要障碍 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 深入、并排分析不同化学修饰对 SM-ASOS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效应。这一点很关键 因为SM-ASO的化学修饰会影响它们的药理性质(例如，细胞 摄取)在不同的细胞类型之间是不同的，从而定义SM-ASO在细胞类型特定物质中的效力。 SM-ASO的化学修饰还规定了潜在的有害影响，如肝脏或肾脏 毒物。因此，这种深入的分析将使选择具有最佳治疗效果的化学物质成为可能 T细胞高效剪接调控的指标(即高效、低毒)。