Aspartoacylase gene transfer in brain/Canavan Disease

这项临床研究的基本原理是先进的基因转移技术可能使患有卡纳万病（CD）的患者受益，卡纳万病是一种具有明确遗传基础的儿童神经退行性疾病。乳糜泻是一种单基因、常染色体隐性遗传病，其中编码天冬氨酸酰化酶（ASPA）的基因有缺陷。功能性酶的缺乏导致底物分子n -乙酰天冬氨酸（NAA）的增加，从而阻止正常的髓鞘形成并导致大脑的海绵状变性。未经治疗的乳糜泻的自然历史是不可逆转的脑损伤和在生命的头十年死亡。目前还没有针对乳糜泻的有效治疗方法，但基因转移有可能阻止或逆转这种致命疾病的病程。我们建议使用重组腺相关病毒（AAV）载体，通过手术将天冬氨酸酰化酶（ASPA）基因直接注入大脑的受影响区域。在之前的一项针对CD的I期研究中，使用第一代载体LPD（由aav为基础的质粒与浓缩剂（硫酸鱼精蛋白）和脂质体制剂（DC-CHOL/DOPE）组成）将手术基因转移到脑CSF空间是安全的。此外，基因转移与局部NAA降低和各种神经功能改善有关。在一些患者中，这些积极的变化是微妙的，而发生的更令人印象深刻的变化是相对短暂的，我们认为这主要是由于载体和递送系统的不足。基因转移技术在过去五年中取得了长足的进步，根据最近的工作，我们已经确定，作为基因传递系统，AAV载体比LPD更有效、更安全，特别是当与肺实质内方法结合使用时。临床前实验表明，AAV作为临床基因转移工具的优势。目前无辅助的AAV包装和纯化方法消除了野生型病毒重组的可能性，并提供了超过1012基因组颗粒/ml的高纯化载体滴度。我们在啮齿动物和灵长类动物中测试的AAV载体包含一个哺乳动物神经启动子元件和额外的后调控元件，这些元件能够驱动高水平的长期转基因表达。含有人天冬氨酸酰化酶cDNA （AAV- aspa）的AAV载体将通过神经外科手术注射到卡纳万病患者的大脑皮层下区域。产前和产后评估将包括非侵入性生化、放射学、电生理、神经学和认知测试。这些结果测量——NAA水平、MRI信号变化、诱发电位和标准化神经发育评估——将被系统记录并进行统计分析。我们期望对该研究数据的细致收集和分析将为更大规模的（II/III期）临床试验提供基础，并且该研究将为未来在人脑中使用病毒载体的所有试验提供有用的参考。

The rationale of this clinical study is that advanced gene transfer techniques may benefit patients suffering from Canavan Disease (CD), a childhood neurodegenerative disorder with a well-defined genetic basis. CD is a monogeneic, autosomal recessive disease in which the gene coding for the enzyme aspartylacylase (ASPA) is defective. The lack of functional enzyme leads to an increase in the substrate molecule, N-acetyl aspartate (NAA), which prevents normal myelination and results in spongiform degeneration of the brain. The natural history of untreated CD is irreversible brain damage and death within the first decade of life. No effective treatment currently exists for CD, but gene transfer has the potential to arrest or reverse the course of this otherwise fatal disease. We propose to surgically administer the aspartylacylase (ASPA) gene directly to affected regions of the brain, using recombinant adeno-associated viral (AAV) vectors. In a previous Phase I study for CD using a first-generation vector known as LPD - comprised of an AAV-based plasmid in conjunction with a condensing agent (protamine sulfate) and a liposomal formulation (DC-CHOL/DOPE) - surgical gene transfer to the CSF space of the brain was shown to be safe. In addition, gene transfer was associated with regionally lowered NAA and various functional neurological improvements. These positive changes were subtle in some patients and the more impressive changes that occurred were relatively transient, which we believe was primarily due to inadequacies of the vector and delivery system. Gene transfer technology has advanced considerably in the last five years and based on more recent work, we have determined that AAV vectors are more efficient and safer than LPD as a gene delivery system, especially when used in conjunction with an intraparenchymal approach. Pre-clinical experiments have been performed which suggest the superiority of AAV as a tool for clinical gene transfer. Current helper-free AAV packaging and purification methods eliminate the possibility of wild-type virus recombination and provide highly purified vector titers exceeding 1012 genomic particles/ml. The AAV vectors that we have tested in rodents and primates contain a mammalian neural promoter element with additional postregulatory elements, which are able to drive high levels of long-term transgene expression. The AAV vector containing human aspartylacylase cDNA (AAV-ASPA) will be neurosurgically injected into defined subcortical regions of the brain in a cohort of patients with Canavan Disease. The pre- and post-delivery assessments will involve non-invasive biochemical, radiological, electrophysiologic, neurological, and cognitive tests. These outcome measures - NAA levels, MRI signal changes, evoked potentials, and standardized neurodevelopmental assessments - will be systematically recorded and statistical analyses will be performed. We anticipate that the meticulous collection and analysis of data from this study will provide the basis for a larger (Phase II/III) clinical trial, and that this study will be useful as a reference for all future trials using viral vectors in the human brain.