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障碍。临床上表现为进行性婴儿帕金森症-肌张力障碍，常被误诊为脑瘫。这些儿童严重残疾，无法控制运动，经历令人痛苦的运动过度和张力障碍姿势。他们逐渐恶化，严重的动作迟缓和肌肉僵硬，称为帕金森氏症。这导致严重的神经残疾，并且无法通过药物或手术治疗，其中三分之一在青春期中期死亡。DTDS是由编码多巴胺转运蛋白（DAT）的SLC 6A 3基因的功能缺失突变引起的。DAT的功能是摄取多巴胺以终止多巴胺神经传递并摄取多巴胺用于再循环，并且在特定的脑区域（中脑和纹状体）中生理性地表达。我们的目标是通过腺相关病毒（AAV）介导的hDAT基因治疗开发一种潜在的治疗DTDS的方法。这将导致正常功能的DAT的表达，以恢复多巴胺稳态，从而改善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显示出巨大的希望。2010年，静脉内递送的腺相关病毒血清型9（AAV 9）拯救了脊髓性肌萎缩症（SMA）小鼠，并在4年内进展到临床试验（NCT 02122952）。SMA 1型是一种严重的运动神经元疾病，受影响的婴儿无法实现坐姿，大多数在20岁时死亡。15名接受治疗的儿童表现出惊人的运动改善，其中12名能够独立坐着，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，称为芳香族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AAV 2-AADC至称为壳核的特定多巴胺能脑区进行治疗。这确立了临床安全性，所有4例儿童均显示运动改善。随后的第二阶段临床试验迄今已治疗了18名儿童。此外，在过去的十年中，超过100名帕金森病患者已经安全地接受了通过立体定向注射递送到纹状体或中脑的AAV 2基因治疗载体。用于帕金森病的AAV 2基因疗法已经递送多巴胺合成基因以增加多巴胺合成，并为AAV 2载体和立体定向注射的安全性提供了良好的临床证据。我们之前进行了DTDS的原理性基因治疗研究的证据，使用DAT-KO小鼠模型。这种DAT-KO小鼠重现了DTDS症状，早期过度运动，59%进入帕金森病阶段，运动迟缓，震颤，体重减轻，到5周龄时存活率降低41%。我们通过脑室内注射向新生DAT-KO小鼠递送AAV 9基因治疗。这挽救了DAT-KO运动表型和存活。然而，通过这种方法，实现了DAT的广泛颅内表达，具有显著的脱靶表达。我们发现，脱靶表达与皮质神经元丢失和反应性星形胶质细胞增生有关。因此，我们的结论是，对于临床应用，这是至关重要的限制hDAT表达多巴胺能神经元。我们的目标是通过将AAV血清型改变为与AAV 9相比具有受限表达谱的AAV 2来提高安全性水平。我们还将载体注射到DTDS中受影响的多巴胺能脑区（中脑和纹状体）。这是走向临床转化的逻辑步骤，其中AAV 2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递送的AAV2.hDAT在成年DAT-KO小鼠中的表达、功效和安全性，作为DTDS基因疗法的概念临床前开发的证据。

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.