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

项目摘要 CLN 1疾病或婴儿神经元蜡样脂褐质沉积症（INCL或婴儿巴滕病）是其中一种 最早发病且进展最快的神经元蜡样脂褐质沉积症（NCL或Batten病）。 CLN 1疾病是由溶酶体酶棕榈酰蛋白硫酯酶-1（PPT 1）缺乏引起的。这 缺乏症对受影响的儿童具有破坏性和迅速发展的影响， 由于没有有效的治疗方法，CLN 1疾病总是致命的。我们有 通过靶向腺相关病毒， 病毒（AAV）介导的基因治疗对受影响最严重的中枢神经系统（CNS）区域， 包括脊髓然而，与其他形式的NCL不同，这种治疗效果仅限于CNS 被转导的区域。因此，成功地将CLN 1疾病的基因治疗转化为临床将是一个巨大的挑战。 对于更大、更复杂的儿童大脑来说是一个重大挑战。为了克服这一障碍，我们 将使用新的CRISPR/Cas9产生的CLN 1 R151 X绵羊模型来完善我们的治疗策略， 评估基因治疗在较大物种中的递送、剂量、安全性和有效性， 适合翻译这些进步。我们最近发表了这些CLN 1 R151 X绵羊显示 明显的CLN 1疾病相关表型。我们的初步数据扩展了这些观察结果，揭示了一个 神经系统疾病的早期发作，以及广泛的组织学和放射学可检测的病理学， 比PPT 1缺陷小鼠更明显。我们还表明，颅内注射AAV 9 表达PPT 1的载体将该酶在绵羊脑中的表达提高到超生理水平。 我们相信CLN 1 R151 X绵羊不仅更准确地模拟人类CLN 1疾病，而且还提供了一种新的方法。 理想的试验场，优化基因治疗的剂量和交付的方式是不可能的， 小鼠我们现在提出在CLN 1 R151 X绵羊中表征这些疾病表型的进展， 为了使用MRI和MRS成像提供疾病进展的详细标志， 这些数据与组织学结果（目的1）。我们还将定义载体剂量和递送的参数 途径，以实现羊脑和脊髓的广泛转导，并提高PPT 1活性， 预测能够产生治疗益处的水平（目标2）。最后，我们将确定 对scAAV 9 - 10的这种优化递送的治疗功效、最小有效剂量、安全性和临床应答。 CCAG-PPT 1对CLN 1 R151 X绵羊的脑和脊髓的作用（目的3）。这些数据将使我们能够改进我们的 基因治疗方法，并使我们进入CREATE-Bio开发计划， 这是对这种毁灭性疾病的第一种有效治疗方法。

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.