Small Oligonucleotides As Therapeutic Agents Of Spinal Muscular Atrophy

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是婴儿死亡的主要遗传原因。最常见的是，SMA由运动神经元和脊髓中全长SMN蛋白（SMN）水平降低所致，这是由于功能性生存运动神经元（SMN 1）等位基因的丧失。该基因（SMN 2）的几乎相同的拷贝无法提供SMA保护，因为在前体mRNA剪接期间SMN 2外显子7的跳跃导致产生截短的SMN。研究人员几乎达成共识，即旨在促进SMN 2外显子7纳入导致全长SMN水平升高的策略将治愈SMA。为了实现这一目标，我们最近报告了一种富含8个核苷酸GC的内含子靶标，通过反义寡核苷酸（阿索）将其隔离完全恢复了SMA患者细胞中的SMN 2外显子7包含。我们的8-mer前导阿索（3UP 8）靶向富含GC序列的反义反应的特异性和效率构成了患者细胞系中短阿索剪接校正的第一个此类报告。短阿索作为治疗剂的无与伦比的益处包括但不限于预期的高特异性、低合成成本、易于修饰和增加的穿过生物屏障递送的机会。SMA目前没有治愈方法。作为SMA治疗的最佳希望之一，我们在此建议开发一种优化的先导阿索变体，可有效纠正SMN 2外显子7剪接，并提高SMA小鼠模型所有组织（包括脑和脊髓）中全长SMN的水平。 在目标1中，我们将优化我们的短阿索用于体内应用。这些将通过一系列定制修饰来实现，包括末端和骨架化学的不同组合。我们将在含有人SMN 2的转基因小鼠中测试修饰的短ASO的功效。我们的初步结果验证了定制修饰改善短阿索在体内剪接校正中的功效的原理证明。我们将进行一系列测试，以确认定制修饰的ASO的体内功效。这些包括但不限于稳定性、剂量反应、脱靶效应、免疫应答和药代动力学特性。我们相信，化学修饰的组合将使我们能够获得优化的电极阿索，其可以有效地递送到包括脑和脊髓在内的所有组织中。还将测试一种替代性的基于脂质纳米颗粒的方法，以在不同组织（包括BBB）中有效地递送我们优化的铅阿索。在目标2中，我们将在轻度和重度SMA小鼠模型中进行实验。我们将确定不同剂量的我们优化的先导阿索对SMA小鼠的表型、生长和发育的影响。特别是，我们寻求改善SMA小鼠的寿命。我们还将在妊娠小鼠中进行实验，以观察产前给药对SMA后代表型和寿命的影响。该提案的成功将为SMA的治疗提供一种基于机制和靶点特异性的药物。) 公共卫生相关性：本项目将开发和测试短反义寡核苷酸在脊髓性肌萎缩症（SMA）小鼠模型中纠正异常剪接的功效。该提案的成功将导致SMA的治疗，SMA是婴儿死亡的主要遗传原因。)

DESCRIPTION (provided by applicant): Spinal Muscular Atrophy (SMA) is a leading genetic cause of infant mortality. Most commonly, SMA results from the reduced levels of full-length SMN protein (SMN) in motor neurons and spinal chord due to the loss of functional Survival Motor Neuron (SMN1) alleles. A nearly identical copy of this gene, SMN2, fails to provide protection from SMA due to production of a truncated SMN because of skipping of SMN2 exon 7 during pre-mRNA splicing. There is a near consensus among researchers that strategies aimed at promotion of SMN2 exon 7 inclusion resulting into the increased levels of full- length SMN would cure SMA. Towards this goal we have recently reported an eight-nucleotide GC- rich intronic target, sequestering of which by an antisense oligonucleotide (ASO) fully restored SMN2 exon 7 inclusion in SMA patient cells. Specificity and efficiency of antisense response by our 8-mer lead ASO (3UP8) targeting GC-rich sequence constitute the first such report of splicing correction by a short ASO in a patient cell line. The unmatched benefits of a short ASO as a therapeutic agent include but not limited to the expected high specificity, low cost of synthesis, ease of modifications and increased chances of delivery across biological barriers. Currently SMA has no cure. As one of the best hopes of SMA therapy, here we propose to develop an optimized variant of our lead ASO that efficiently corrects SMN2 exon 7 splicing and raise the levels of full-length SMN in all tissues including brain and spinal cord of mice models of SMA. In Aim 1, we will optimize our short ASO for application in vivo. These will be accomplished by a series of custom modifications including different combinations of the terminal and backbone chemistries. We will test the efficacy of modified short ASOs in transgenic mice containing human SMN2. Our initial results validate the proof-of-principle that custom modifications improve the efficacy of a short ASO in splicing correction in vivo. We will run a series of tests to confirm the in vivo efficacy of the custom-modified ASOs. These include but not limited to stability, dose response, off-target effects, immune response and pharmacokinetic properties. We believe that a combination of chemical modifications will allow us to obtain an optimized lead ASO that could be effectively delivered in all tissues including brain and spinal cord. Will also test an alternatively lipid-nanoparticle-based approach to efficiently deliver our optimized lead ASO in different tissues including across BBB. In Aim 2, we will perform experiments in mild as well as severe SMA mouse models. We will determine the effect of different doses of our optimized lead ASO on the phenotype, growth and development of SMA mice. In particular, we seek to improve the longevity of SMA mice. We will also conduct experiments in pregnant mice to see the effect of pre-natal drug delivery on the phenotype and longevity of SMA offspring. The success of this proposal will provide a mechanism-based and target- specific drug for the treatment of SMA. ) PUBLIC HEALTH RELEVANCE: This project will develop and test the efficacy of short antisense oligonucleotides for correction of aberrant splicing in mice models of spinal muscular atrophy (SMA). The success of this proposal will lead to the treatment of SMA, a leading genetic cause of infant mortality. )