Development of kinetin as a treatment for familial dysautonomia

描述（由申请人提供）：家族性自主神经异常（FD）是一种由IKBKAP基因剪接突变引起的遗传性感觉和自主神经病变。该突变导致IKBKAP mRNA外显子20的可变跳变，从而导致IKAP蛋白的组织特异性减少。FD患者的组织分析显示，神经元组织中显着更多的外显子跳跃，因此IKAP水平较低。IKAP是人类伸长复合体的一个成员，它是基因子集有效转录伸长所必需的。研究表明，IKAP的缺失导致伸长依赖基因的表达减少，从而导致细胞迁移缺陷，这可能解释了FD中所见的神经发育缺陷。尽管FD是隐性的，但我们已经证明患者保留了制造正常mRNA和蛋白质的能力。这一发现为开发旨在通过剪接修饰提高细胞IKAP水平的疗法提供了一种令人兴奋的直接方法。作为NINDS赞助的神经代药物筛选联盟的一部分，我们发现用kinetin（一种植物细胞分裂素）处理培养的FD细胞，增强了外显子20的包裹性，并显着增加了FD细胞中野生型IKBKAP mRNA和IKAP蛋白的数量。对患者细胞系的分析表明，在动素治疗的三天内，正常的IKAP水平与正常细胞的水平相当。为了开发动素作为FD的治疗方法，我们将FD突变插入人BAC并创建了几个转基因小鼠系。重要的是，这些小鼠系中的转基因表达表明，异常FD剪接的人类模式被保留了下来。此外，对几种小鼠组织的分析表明，大脑中的异常剪接明显更严重，完全模拟了我们在FD患者中观察到的情况。这些小鼠将使我们能够在体内测试动素，以确定该药物是否可以增加正常人的IKAP水平。我们最近还表明，IKBKAP在小鼠中的完全丧失会导致胚胎死亡。通过转基因拯救基因敲除小鼠，我们的目标是建立一个准确的FD表型模型，以评估动素的疗效。尽管FD是一种发育障碍，但患者一生都受到持续、剧烈的神经元变性的困扰。在生命早期有效地增加IKAP水平可以支持神经元存活，预防或延缓青少年患者出现的衰弱性步态、感觉和认知能力下降。家族性自主神经异常是一种严重的神经发育疾病，由IKBKAP基因突变引起。这种突变导致所有组织中的蛋白质表达减少，但这种减少是可变的，在神经元组织中最严重。我们已经确定了一种药物，kinetin，可以增加患者细胞中的IKAP蛋白，我们的目标是开发这种药物作为FD患者的治疗方法。这笔拨款的目的是测试这种药物，看看它是否在患有这种疾病的小鼠模型中起作用。

DESCRIPTION (provided by applicant): Familial dysautonomia (FD) is a hereditary sensory and autonomic neuropathy that is caused by a splice mutation in the IKBKAP gene. The mutation results in variable skipping of exon 20 in IKBKAP mRNA, which leads to a tissue-specific reduction of IKAP protein. Analysis of tissue from FD patients shows significantly more exon skipping in neuronal tissue, and therefore lower IKAP levels. IKAP is a member of the human Elongator complex, which is required for efficient transcriptional elongation of a subset of genes. Studies have shown that depletion of IKAP leads to a defect in cell migration due to reduced expression of Elongator dependent genes, which might explain the neurodevelopmental defects seen in FD. Despite the fact that FD is recessive, we have shown that patients retain the capacity to make both normal mRNA and protein. This discovery offers an exciting, direct approach towards the development of therapies aimed at increasing levels of cellular IKAP via splicing modification. As part of the NINDS sponsored Neurogeneration Drug Screening Consortium, we found that treatment of cultured FD cells with kinetin, a plant cytokinin, enhances exon 20 inclusion and dramatically increases the amount of wild-type IKBKAP mRNA and IKAP protein in FD cells. Analysis of patient cell lines shows that within three days of kinetin treatment the level of normal IKAP is equivalent to that seen in normal cells. In order to develop kinetin as a therapy for FD, we have inserted the FD mutation into a human BAC and created several transgenic mouse lines. Importantly, transgene expression in these mouse lines shows that the human pattern of aberrant FD splicing is preserved. Moreover, analysis of several mouse tissues shows that aberrant splicing is significantly worse in the brain, modeling perfectly what we observe in FD patients. These mice will enable us to test kinetin in vivo to determine if the drug can increase levels of normal human IKAP. We have also recently shown that a complete loss of IKBKAP in mice leads to embryonic lethality. By rescuing the knock-out mouse with the transgene, we aim to create an accurate phenotypic model of FD for evaluating the efficacy of kinetin. Despite the fact that FD is a developmental disorder, patients are plagued by continued, drastic neuronal degeneration throughout life. Effectively increasing IKAP levels early in life may support neuronal survival and prevent or delay the debilitating gait, sensory, and cognitive decline seen in teenage patients.Familial dysautonomia is a severe neurodevelopmental disease that is caused by a mutation in the IKBKAP gene. This mutations leads to reduced protein expression in all tissues, but this reduction is variable and is most severe in neuronal tissue. We have identified a drug, kinetin, that can increase IKAP protein in patient cells and our goal is to develop this drug as a therapy for FD patients. The purpose of this grant is to test this drug to see if it works in mouse models of the disease.