Small Oligonucleotides As Therapeutic Agents Of Spinal Muscular Atrophy

描述(申请人提供)：脊髓性肌萎缩症(SMA)是导致婴儿死亡的主要遗传原因。最常见的SMA是由于功能性存活运动神经元(SMN1)等位基因的丢失而导致运动神经元和脊髓中全长SMN蛋白(SMN)水平的降低。该基因的一个几乎相同的拷贝SMN2，由于在前mRNA剪接过程中跳过SMN2外显子7而产生截短的SMN，因此无法提供对SMA的保护。研究人员几乎一致认为，旨在促进SMN2外显子7包含导致全长SMN水平增加的策略将治愈SMA。为了实现这一目标，我们最近报道了一个富含GC的八核苷酸内含子靶点，通过反义寡核苷酸(ASO)完全恢复SMA患者细胞中SMN2外显子7的包涵体。我们的8-聚体先导ASO(3UP8)针对富含GC序列的反义反应的特异性和有效性构成了第一个通过短ASO在患者细胞系中进行剪接纠正的报告。短ASO作为治疗剂的无与伦比的好处包括但不限于预期的高特异性、低合成成本、易于修饰和增加跨越生物屏障的机会。目前，SMA还没有治愈方法。作为SMA治疗的最大希望之一，我们建议开发我们的Lead ASO的优化变体，该变体可以有效地纠正SMN2外显子7的剪接，并提高SMA模型小鼠包括脑和脊髓在内的所有组织中全长SMN的水平。在目标1中，我们将优化我们的短ASO用于体内应用。这些将通过一系列定制修改来实现，包括终端和主干化学物质的不同组合。我们将在含有人SMN2的转基因小鼠身上测试改良的短ASO的效果。我们的初步结果验证了定制修改在体内提高短ASO在剪接纠正方面的有效性的原则证明。我们将进行一系列测试，以确认定制修改的ASOS的体内疗效。这些包括但不限于稳定性、剂量反应、靶外效应、免疫反应和药代动力学特性。我们相信，化学修饰的组合将使我们能够获得优化的铅ASO，该ASO可以在包括大脑和脊髓在内的所有组织中有效地传递。还将测试一种替代的基于脂质纳米颗粒的方法，以在不同的组织中高效地传递我们优化的ASO铅，包括跨血脑屏障。在目标2中，我们将在轻度和重度SMA小鼠模型上进行实验。我们将确定不同剂量的我们优化的铅ASO对SMA小鼠的表型、生长和发育的影响。特别是，我们寻求提高SMA小鼠的寿命。我们还将在怀孕的小鼠身上进行实验，看看产前给药对SMA后代的表型和寿命的影响。这一提议的成功将为SMA的治疗提供一种基于机制和靶向性的药物。)

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. )