Characterizing and testing the efficacy of AAV-mediated gene therapy in a novel CRISPR/Cas9 generated sheep model of Cln1 disease.

项目总结 CLN1病或婴儿神经性干酪样脂褐质沉着症(包括婴儿巴顿病)是 起病最早、进展最快的神经性蜡样脂褐素沉着症(NCL或Batten病)。 CLN1疾病是由溶酶体酶棕榈酰基蛋白硫酯酶-1(PPT1)缺乏引起的。这 缺乏症会对受影响的儿童产生破坏性的、快速发展的影响，这种影响始于第一个 由于没有有效的治疗方法，CLN1疾病总是致命的。我们有 能够通过靶向腺相关基因显著改善PPT1缺陷小鼠的治疗结果 病毒(AAV)介导的对受影响最严重的中枢神经系统(CNS)区域的基因治疗， 包括脊髓。然而，与其他形式的NCL不同，这种治疗作用仅限于中枢神经系统 被转换的区域。因此，成功地将CLN1疾病的基因疗法转化为临床将 对一个孩子更大更复杂的大脑来说是一个重大的挑战。为了克服这一障碍，我们 将使用CRISPR/Cas9生成的CLN1 R151X绵羊模型来完善我们的治疗策略， 评估基因疗法在较大物种中的传递、剂量、安全性和有效性，这是理想的 适合翻译这些进步。我们最近发布了这些CLN1 R151X绵羊展示 明显的CLN1疾病相关表型。我们的初步数据扩展了这些观察，揭示了 较早发病的神经系统疾病，以及广泛的组织学和放射学可检测到的病理 比PPT1基因缺陷的小鼠更明显。我们还表明，脑部注射AAV9 表达PPT1的载体将该酶在绵羊脑中的表达提高到超生理水平。 我们认为CLN1 R151X绵羊不仅可以更准确地模拟人类CLN1疾病，而且还提供了一种 理想的试验场，用于优化基因疗法的剂量和传递，这种方式在 老鼠。我们现在建议表征这些疾病表型在CLN1 R151X绵羊中的进展， 以便使用MRI和MRS成像提供疾病进展的详细标志，相互关联 这些数据与组织学结果(目标1)相一致。我们还将定义载体投放和投放的参数 实现绵羊大脑和脊髓的广泛转导，并将PPT1活性提高到 预测能够产生治疗效益的水平(目标2)。最后，我们将确定 对scAAV9-优化给药的疗效、最小有效剂量、安全性和临床反应 CCAG-PPT1对CLN1 R151X绵羊脑和脊髓的作用(目的3)。这些数据将使我们能够改进我们的 基因治疗方法，并为我们进入Create-Bio临床开发计划做好准备 翻译了第一个有效治疗这种毁灭性疾病的方法。

PROJECT SUMMARY CLN1 disease or Infantile Neuronal Ceroid Lipofuscinosis (INCL or Infantile Batten disease) is one of the earliest onset and most rapidly progressing forms of neuronal ceroid lipofuscinosis (NCL or Batten disease). CLN1 disease is caused by deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). This deficiency has a devastating and rapidly progressing effect upon affected children that starts within the first year of life, and because there is no effective therapy available CLN1 disease is always fatal. We have been able to dramatically improve therapeutic outcomes in PPT1-deficient mice by targeting adeno-associated viral (AAV)-mediated gene therapy to the central nervous system (CNS) regions that are most affected, including the spinal cord. However, unlike other forms of NCL, such therapeutic effects are limited to the CNS regions that are transduced. As such, successfully translating gene therapy for CLN1 disease into the clinic will be a significant challenge in the much larger and more complex brain of a child. To overcome this obstacle, we shall use a novel CRISPR/Cas9 generated CLN1 R151X sheep model to refine our therapeutic strategy, assessing the delivery, dosing, safety and efficacy of gene therapy in a larger species that is ideally suited for translating these advances. We recently published that these CLN1 R151X sheep display pronounced CLN1 disease-relevant phenotypes. Our preliminary data extend these observations, revealing an earlier onset of neurologic disease, and widespread histologically and radiologically detectable pathology that is more pronounced than in PPT1-deficient mice. We have also shown that an intracranial injection of an AAV9 vector expressing PPT1 raises expression of this enzyme in the brain of sheep to supraphysiological levels. We believe CLN1 R151X sheep not only more accurately model human CLN1 disease, but also provide an ideal testing ground for optimizing the dosing and delivery of gene therapy in a fashion that is not possible in mice. We now propose to characterize the progression of these disease phenotypes in CLN1 R151X sheep, in order to provide detailed landmarks of disease progression using both MRI and MRS imaging, correlating these data with histological findings (Aim 1). We will also define the parameters of vector dosing and delivery routes to achieve widespread transduction of the sheep brain and spinal cord, and elevate PPT1 activity to levels predicted to be capable of producing therapeutic benefit (Aim 2). Finally, we shall determine the therapeutic efficacy, minimum effective dose, safety and clinical response to this optimized delivery of scAAV9- CCAG-PPT1 to the brain and spinal cord of CLN1 R151X sheep (Aim 3). These data will allow us to refine our gene therapy approach, and position us for entry into the CREATE-Bio development program towards clinical translation of the first effective treatment of this devastating disease.