Peptide conjugated oligonucleotides for splice switching therapy of Spinal Muscular Atrophy

脊髓性肌萎缩症（SMA）是婴儿死亡的主要遗传原因，由SMN 1基因功能丧失引起。SMN 1突变导致运动神经元变性，伴有外周表现，包括骨骼肌萎缩。SMA是罕见的，发病率约为1：10，000活产;然而，每35个未受影响的个体中就有1个可能是该疾病的携带者。大多数受影响的SMA婴儿通常患有严重形式的疾病，平均预期寿命约为2岁。然而，病情较轻的儿童可以存活超过2年，但生活质量较差。SMA严重程度与患者产生的功能性SMN蛋白水平直接相关。与SMN 1密切相关的基因是SMN 2，尽管该基因通常仅产生约10%的全功能SMN蛋白。然而，一些SMA患者具有额外的SMN 2基因拷贝，因此可以产生更多功能性SMN蛋白。这减轻了疾病的严重程度，并且此类患者通常具有较轻的病程。大多数SMN 2基因产物无功能，因为该基因通过选择性剪接产生两种不同的mRNA，即大多数mRNA缺乏外显子7，仅产生部分功能蛋白。已经开发了一种高度新颖的治疗方法，其作用于SMN 2 mRNA，影响外显子7的包含，以产生功能性SMN蛋白。该治疗使用剪接转换寡核苷酸（SSO），其是与SMN 2 mRNA特异性结合的化学修饰的DNA片段。近年来，SSO疗法在治疗杜氏肌营养不良症（DMD）方面显示出巨大的潜力，目前正处于高级临床试验阶段。SSO疗法有可能对SMA产生更大的影响，因为它有可能治疗近100%的受影响SMA患者。成功开发SMA SSO疗法的主要挑战是将SSO药物递送到严重受影响的组织，包括运动神经元和骨骼肌。递送至脊髓通常需要侵入性方法，而靶向重要的外周组织（如骨骼肌）是低效的，并且通常需要高SSO浓度。目前最先进的技术，由伊希斯制药公司领导，涉及一项临床试验，在该试验中，SSO通过注射到脊髓周围的液体中对SMA患者进行侵入性给药，因此不治疗外周组织。通过简单的静脉注射侵入性地治疗外周组织，并且也充分穿透到脑和脊髓中以治疗神经系统疾病。疾病的组成部分。我们已经开发了先进的SSO，其与短蛋白片段（肽）偶联以促进改善SSO向外周组织的递送并提供对脑和脊髓的渗透。关键的初始步骤是选择最佳肽。在我们现有的肽平台的基础上，我们还将研究一些具有高脑递送潜力的其他肽。一旦选择了候选肽和SSO，我们将对SSO药物的组织分布和组织浓度以及最佳剂量要求进行广泛的研究。这些信息将使我们能够设计多剂量、长期的SSO治疗方案，以全身治疗所有与SMA相关的组织，并且我们将在SMA动物模型中严格评估这一点。我们之前在治疗DMD方面的成功以及与SSO药物相关的肽化学经验使我们在该项目中取得了独特的成功。成功之后，将采取步骤进行SSO先导药物的临床开发。

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, arising from loss-of-function of the SMN1 gene. Mutations in SMN1 result in motor neuron degeneration, accompanied by peripheral manifestations including skeletal muscle atrophy. SMA is rare with an incidence of ~1:10,000 live births; however 1 in every 35 unaffected individuals is likely to be a carrier for the disease. Most affected SMA infants typically have a severe form of the disease with a mean life expectancy of ~2 years in age. However children with less severe disease can survive beyond 2 years but with poor quality of life. SMA severity relates directly to the level of functional SMN protein that a patient produces. A closely related gene to SMN1 is SMN2, although this gene typically only produces ~10% of fully functional SMN protein. However some SMA patients have additional copies of the SMN2 gene, and hence can produce more functional SMN protein. This mitigates disease severity and such patients typically have a milder disease course. Most SMN2 gene product is not functional because the gene generates two distinct mRNAs via alternative splicing i.e. most of the mRNA lacks exon 7 and generates only partially functional protein. A HIGHLY NOVEL TREATMENT has been developed which acts on SMN2 mRNA to influence inclusion of exon 7 to generate functional SMN protein. This treatment utilizes splice switching oligonucleotides (SSOs), which are chemically modified DNA fragments that bind specifically to the SMN2 mRNA. SSO therapy has shown great potential in recent years for the disorder Duchenne muscular dystrophy (DMD) where they are now in advanced clinical trials. An SSO therapy has potential for even greater impact in SMA because it has potential to treat nearly 100% of affected SMA patients.The major challenge to successful development of an SSO therapy for SMA is delivery of the SSO drug to critically affected tissues including motor neurons and skeletal muscle. Delivery to the spinal cord typically requires invasive methods, while targeting important peripheral tissues, such as skeletal muscle, is inefficient and often requires high SSO concentrations. The current state-of-the-art, led by Isis Pharmaceuticals, involves a clinical trial in which SSO is administered invasively to SMA patients via injection into the fluid surrounding the spinal cord and which therefore does not treat peripheral tissues.Our aim is to develop an advanced SSO that can be administered non-invasively through a simple intravenous injection to treat peripheral tissues and also penetrates sufficiently well into the brain and spinal cord to treat the neurological component of the disease. We have developed advanced SSOs to which are coupled short protein fragments (peptides) to facilitate improved SSO delivery to peripheral tissues and provide penetration into the brain and spinal cord. A key initial step is to select the optimal peptide. Building on our existing peptide platform, we will also study a number of additional peptides with high brain delivery potential. Once a candidate peptide and SSO has been selected we will conduct extensive studies into the tissue distribution and tissue concentration of the SSO drug and also into the optimal dosing requirements. This information will allow us to design a multi-dose, long-term SSO therapy regimen to systemically treat all tissues associated with SMA, and we will evaluate this rigorously in an animal model of SMA. Our previous success with treating DMD and experience with peptide chemistry in relation to SSO drugs makes us uniquely well-placed for success in this project. Success will be followed by steps towards clinical development of the lead SSO drug.