Oligonucleotides that Modulate AMPA Receptor Alternative Splicing as Drug Candida

描述（由申请人提供）： 肌萎缩侧索硬化症（ALS）的特征在于运动皮层、脑干和脊髓中的运动神经元（MN）的进行性和选择性丧失，导致患者通常在诊断后2-5年死亡。CNS中谷氨酸介导的兴奋性毒性已被认为是ALS中MN细胞死亡的主要原因。据认为，MN对谷氨酸介导的兴奋性毒性的脆弱性是由于其高密度的Ca 2+渗透性离子通道和其处理过量Ca 2+内流的能力降低的组合，这是神经变性的触发因素。为什么ALS患者的MN特别容易受到兴奋性毒性的影响仍然未知，但几乎普遍接受兴奋性毒性导致ALS患者MN的选择性死亡。因此，开发降低CNS兴奋性活动期间Ca 2+内流的药物是寻求ALS治疗方法的关键焦点。离子型AMPA受体是谷氨酸激活的离子通道，是MN中Ca 2+内流的关键介质。AMPA受体由称为GluR 1 -4的四个亚基组成。所有四种GluR的两种选择性剪接变体称为“翻转”和“翻转”，通常在CNS中表达，并且已知调节AMPA通道电导。含有GluR-翻转同种型的AMPA通道是“较高增益”通道，其具有比含有“翻转”变体的“较低增益”AMPA通道更大的电流幅度和/或对谷氨酸脱敏更大的抗性。因此，当GluRs的翻转/翻转比率升高时，MN中的AMPA受体显示增强的兴奋活性和更大的Ca 2+内流。我们最近开发了几种新的化学修饰的RNA寡核苷酸，称为剪接调节寡聚体（SMO），它有效地和特异性地调节GluR选择性剪接，以减少各种GluR亚型的翻转/翻转比。尽管SMO不穿过血脑屏障，但在直接递送到CSF中后，它们广泛分布在整个CNS中，并且具有容易穿过膜并进入CNS中细胞核的非凡能力，其中它们的剪接调节活性持续数月。在这里，我们提出了一套翻译实验，以验证我们的SMO作为治疗ALS的疗法的潜力。首先，我们将比较我们的电极导线SMO（LSP-GR 3）在降低整个CNS中GluR 3翻转表达方面的有效性，使用（1）连续ICV给药和（2）单次推注到脊柱腰囊中（目的1）。然后，我们将使用目标1中确定的最佳递送方式，并评估我们的两种铅SMO改善ALS小鼠运动功能和延长寿命的功效（目标2）。 公共卫生相关性： 该提案旨在提供LifeSplice Pharma，LLC开发的新型候选药物在改善ALS小鼠病理学方面有效的“原理证明”。这些研究将导致正式的毒理学分析，并最终获得IND申请和FDA批准用于ALS患者的临床试验。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is characterized by the progressive and selective loss of motor neurons (MNs) in the motor cortex, brainstem, and spinal cord, resulting in death of patients typically 2-5 years after diagnosis. Glutamate-mediated excitotoxicity in the CNS has been implicated as a major contributor to MN cell death in ALS. It is thought that the vulnerability of MNs to glutamate-mediated excitotoxicity is due to a combination of their high density of Ca2+ permeable ion channels, and their diminished capacity to handle excessive Ca2+ influx, which serves as a trigger for neurodegeneration. Why MNs of ALS patients are especially vulnerable to excitotoxicity remains unknown, but there is nearly universal acceptance that excitotoxicity contributes to selective death on MNs in ALS patients. Therefore, the development of drugs which lower Ca2+ influx during excitatory activity in the CNS, is a crucial focal point in the pursuit for therapeutics for ALS. Ionotropic AMPA receptors are glutamate-activated ion channels that are key mediators of Ca2+ influx in MNs. AMPA receptors are composed of four subunits termed GluR1-4. Two alternatively spliced variants of all four GluRs called "flip" and "flop" are normally expressed in the CNS and are known to modulate AMPA channel conductance. AMPA channels containing GluR-flip isoforms are 'higher gain' channels, which have greater current amplitudes and/or greater resistant to glutamate-desensitization than the 'lower gain' AMPA channels containing "flop" variants. Thus, when the flip/flop ratios of GluRs are elevated, AMPA receptors in MNs show enhanced excitatory activity, and greater Ca2+ influx. We recently developed several novel chemically-modified RNA oligonucleotides, called splice modulating oligomers (SMOs), which potently and specifically modulate GluR alternative splicing to reduce the flip/flop ratio of various GluR isoforms. Although SMOs do not cross the blood-brain-barrier, after direct delivery into the CSF they are broadly distributed throughout the CNS and have the extraordinary capacity to readily to cross the membrane and enter the nuclei of cells in the CNS, where their splice modulating activity persists for months. Here we propose a translational set of experiments to validate the potential of our SMOs as therapeutics for treating ALS. First, we will compare the efficacy of our lead SMO (LSP-GR3) in reducing GluR3-flip expression throughout the CNS, using (1) continuous ICV delivery, and (2) single bolus injection into the lumbar sac of the spinal column (Aim 1). We will then use the best delivery modality determined in Aim 1 and evaluate the efficacy of our two lead SMOs to improve motor function, and increase lifespan in ALS mice (Aim 2). PUBLIC HEALTH RELEVANCE: This proposal is designed to provide the 'proof of principle' that novel drug candidates developed by LifeSplice Pharma, LLC are efficacious at ameliorating pathology in ALS mice. These studies will lead to formal toxicology analysis, and ultimately an IND application and FDA approval for clinical trials in ALS patients.