Gene therapy for Childhood Parkinsonism: Dopamine transporter deficiency syndrome

多巴胺转运蛋白缺乏综合征（DTDS）是一种毁灭性的儿童神经递质疾病。临床表现为进行性小儿帕金森病-肌张力障碍，常误诊为脑瘫。这些儿童严重残疾，无法控制运动，出现令人痛苦的运动亢进和肌张力障碍。它们会随着严重的运动缓慢和肌肉僵硬而逐渐恶化，称为帕金森病。这会导致严重的神经残疾，无法用药物或手术治疗，三分之一的人在青春期中期死亡。DTDS是由编码多巴胺转运体（DAT）的SLC6A3基因的功能突变丧失引起的。DAT的功能是摄取多巴胺，终止多巴胺神经传递和摄取多巴胺进行再循环，并在特定脑区（中脑和纹状体）进行生理表达。对于这种无法治愈的儿童神经系统疾病，医学上需要开发有效的治疗方法。我们的目标是通过提供腺相关病毒（AAV）介导的hDAT基因治疗来开发一种潜在的治疗DTDS的方法。这将导致正常功能DAT的表达，以恢复多巴胺稳态，改善DTDS儿童的运动功能、健康和神经功能障碍。儿童神经系统疾病的基因治疗正显示出巨大的希望。腺相关病毒血清型9 （AAV9）于2010年静脉注射拯救脊髓性肌萎缩症（SMA）小鼠，并在4年内进入临床试验（NCT02122952）。1型肌萎缩萎缩症是一种严重的运动神经元障碍，患病的婴儿无法坐姿，大多数在2岁前死亡。15名接受治疗的儿童表现出惊人的运动改善，其中12名能够坐下来，一些能够独立行走。基因疗法用于治疗另一种儿童神经递质疾病，称为芳香l -氨基酸脱羧酶（AADC）缺乏症，临床上与DTDS相似。最初，4名儿童通过直接（立体定向）注射AAV2-AADC到特定的多巴胺能脑区，称为壳核。这建立了临床安全性，所有4名儿童均表现出运动改善。到目前为止，随后的II期临床试验已经治疗了18名儿童。此外，在过去的十年中，超过100名帕金森病患者安全地接受了立体定向注射到纹状体或中脑的AAV2基因治疗载体。AAV2基因治疗帕金森病，传递多巴胺合成基因，增加多巴胺合成，为AAV2载体和立体定向注射的安全性提供了良好的临床证据。我们之前使用DAT-KO小鼠模型进行了DTDS的原理证明基因治疗研究。这只DAT-KO小鼠再现了早期运动过度的DTDS症状，59%的小鼠在5周龄时进入帕金森期，伴有运动迟缓、震颤、体重减轻，存活率降低41%。我们将AAV9基因疗法注入新生DAT-KO小鼠脑室内。这挽救了DAT-KO运动表型和存活。然而，通过这种方法，可以实现DAT在颅内的广泛表达，并有明显的脱靶表达。我们发现脱靶表达与皮层的神经元丢失和反应性星形胶质细胞增生有关。因此，我们得出结论，在临床应用中，限制hDAT在多巴胺能神经元中的表达是至关重要的。我们的目标是通过将AAV血清型改为与AAV9相比表达谱受限的AAV2来提高安全性。我们还将把载体注入DTDS（中脑和纹状体）中受影响的多巴胺能脑区域。这是朝着临床翻译迈出的合乎逻辑的一步，AAV2和立体定向注射方法已经建立了临床安全性。我们将评估立体定向给药AAV2的表达、疗效和安全性。成年DAT-KO小鼠的hDAT作为DTDS基因治疗概念临床前开发的证明。

Dopamine transporter deficiency syndrome (DTDS) is a devastating childhood neurotransmitter disorder. It clinically presents with progressive infantile parkinsonism-dystonia and is frequently misdiagnosed as cerebral palsy. The children are severely disabled with inability to control movement experiencing distressing hyperkinesia and dystonic postures. They progressively worsen with severe slowing of movements and muscle rigidity, termed parkinsonism. This results in severe neurodisability and is untreatable with medication or surgery, with a third dying by mid-adolescence. DTDS results from loss of function mutations the SLC6A3 gene coding for the Dopamine transporter (DAT). DAT functions to uptake dopamine to terminate dopamine neurotransmission and uptake dopamine for recycling and is physiologically expressed in specific brain regions (midbrain and striatum).There is medical need to develop impactful treatment for this untreatable childhood neurological disorders. We aim to develop a potential cure for DTDS by delivering Adeno associated virus (AAV) mediated hDAT gene therapy. This will result in expression of normally functioning DAT to restore dopamine homeostasis to improve motor function, health and neurodisability of DTDS children. Gene therapy for childhood neurological disorders is showing great promise. Adeno associated virus serotype 9 (AAV9) delivered intravenously rescued the Spinal muscular atrophy (SMA) mouse in 2010 and progressed to clinical trial within 4 years (NCT02122952). SMA type 1 is severe motor neuron disorder whereby affected babies do not achieve sitting and most die by 2years. Fifteen treated children have shown striking motor improvements, with 12 able to sit and some walking independently. Gene therapy is used to treat another childhood neurotransmitter disorder called Aromatic L-amino acid decarboxylase (AADC) deficiency, that is clinically similar to DTDS. Initially 4 children were treated by direct (stereotactic) injection of AAV2-AADC to a specific dopaminergic brain region called the putamen. This established clinical safety and all 4 children showed motor improvement. A subsequent phase II clinical trial has treated 18 children to date. Furthermore over 100 patients with Parkinson's disease, have safely received AAV2 gene therapy vectors delivered by stereotactic injection to the striatum or midbrain over the last decade. AAV2 gene therapies for Parkinson's disease have delivered dopamine synthesis genes to increase dopamine synthesis and provide good clinical evidence for the safety of AAV2 vectors and stereotactic injection. We previously undertook proof of principle gene therapy study for DTDS using the DAT-KO mouse model. This DAT-KO mouse recapitulates DTDS symptoms with early hyperlocomotion, 59% entering a Parkinsonian phase with bradykinesia, tremor, weight loss and reduced survival of 41%, by 5 weeks old. We delivered AAV9 gene therapy to neonatal DAT-KO mice by injection to the intracerebroventrical. This rescued the DAT-KO motor phenotype and survival. However with this approach, widespread intracranial expression of DAT was achieved, with significant off-target expression. We identified that off-target expression was associated with neuronal loss and reactive astrogliosis in the cortex. We therefore concluded that for clinical application, it is paramount to restrict hDAT expression to dopaminergic neurons. We aim to improve safety levels by changing AAV serotype to AAV2 that has a restricted expression profile compared to AAV9. We will also inject the vector into the dopaminergic brain regions affected in DTDS (midbrain and striatum). This is a logical step towards clinical translation whereby AAV2 and stereotactic injection methods have established clinical safety. We will evaluate expression, efficacy, and safety of stereotactically delivered AAV2.hDAT in adult DAT-KO mice as proof of concept preclinical development of gene therapy for DTDS.