Development of kinetin as a treatment for familial dysautonomia

描述（申请人提供）：家族性自主神经功能障碍（FD）是一种遗传性感觉和自主神经病变，由IKBKAP基因剪接突变引起。该突变导致IKBKAP mRNA中外显子20的可变跳跃，这导致IKAP蛋白的组织特异性减少。对FD患者组织的分析显示，神经元组织中的外显子跳跃显著更多，因此IKAP水平较低。IKAP是人类延伸子复合体的成员，其是基因子集的有效转录延伸所需的。研究表明，IKAP的缺失导致细胞迁移缺陷，这是由于延长子依赖性基因的表达减少，这可能解释FD中观察到的神经发育缺陷。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、直接的方法。作为NINDS赞助的神经生成药物筛选联盟的一部分，我们发现用激动素（一种植物细胞分裂素）处理培养的FD细胞，可增强FD细胞中20号外显子的包含，并显著增加野生型IKBKAP mRNA和IKAP蛋白的量。对患者细胞系的分析表明，在激动素治疗的三天内，正常IKAP的水平与正常细胞中所见的水平相当。为了开发激动素作为FD的治疗方法，我们将FD突变插入人BAC中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显示异常FD剪接的人类模式被保留。此外，对几种小鼠组织的分析表明，异常剪接在大脑中明显更糟，完美地模拟了我们在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激动素，以确定药物是否可以增加正常人类IKAP的水平。我们最近还表明，IKBKAP在小鼠中的完全缺失会导致胚胎死亡。通过用转基因挽救基因敲除小鼠，我们的目标是建立一个准确的FD表型模型，用于评估激动素的疗效。尽管FD是一种发育障碍，但患者一生中仍受到持续、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的存活，预防或延迟在青少年患者中观察到的衰弱性步态、感觉和认知下降。家族性自主神经功能障碍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蛋白质表达减少，但这种减少是可变的，并且在神经元组织中最严重。我们已经确定了一种药物激动素，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。这项资助的目的是测试这种药物，看看它是否在这种疾病的小鼠模型中有效。

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.