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，由于功能性存活运动神经元（SMN1）等位基因的缺失，运动神经元和脊髓中全长SMN蛋白（SMN）水平降低。该基因的一个几乎相同的拷贝SMN2，由于在mrna前剪接过程中SMN2外显子7的跳跃而产生截短的SMN，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的包含从而增加全长SMN水平的策略可以治愈SMA。为了实现这一目标，我们最近报道了一个富含8个核苷酸GC的内含子靶标，通过反义寡核苷酸（ASO）将其隔离，完全恢复了SMA患者细胞中SMN2外显子7的包涵。我们的靶向GC-rich序列的8-mer先导ASO （3UP8）反义反应的特异性和效率是首个在患者细胞系中使用短ASO进行剪接纠正的报道。短ASO作为治疗药物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，我们建议开发一种优化的ASO变体，有效地纠正SMN2外显子7剪接，并提高SMA小鼠模型中所有组织（包括脑和脊髓）中全长SMN的水平。在Aim 1中，我们将优化我们的短ASO以在体内应用。这些将通过一系列定制的修改来完成，包括终端和主链化学的不同组合。我们将在含有人SMN2的转基因小鼠中测试修饰的短ASOs的功效。我们的初步结果验证了自定义修饰提高短ASO在体内剪接校正效果的原理证明。我们将进行一系列试验，以确认定制修改的ASOs的体内功效。这些包括但不限于稳定性、剂量反应、脱靶效应、免疫反应和药代动力学性质。我们相信，化学修饰的结合将使我们获得一种优化的ASO铅，可以有效地输送到包括脑和脊髓在内的所有组织中。我们还将测试一种基于脂质纳米粒子的替代方法，以有效地将我们优化的ASO导联输送到不同的组织中，包括血脑屏障。在Aim 2中，我们将在轻度和重度SMA小鼠模型中进行实验。我们将测定不同剂量优化后的ASO铅对SMA小鼠表型、生长发育的影响。特别是，我们寻求提高SMA小鼠的寿命。我们还将在怀孕小鼠中进行实验，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向特异性的药物。

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. )