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Canavan病(CD)的患者受益，CD是一种儿童神经退行性疾病，具有明确的遗传基础。CD是一种单基因常染色体隐性遗传病，其天冬氨酸氨基转移酶(ASPA)基因编码缺陷。缺乏功能酶会导致底物分子N-乙酰天冬氨酸(NAA)的增加，这会阻止正常的髓鞘形成，并导致大脑海绵状变性。未经治疗的CD的自然历史是不可逆转的脑损伤和生命第一个十年内的死亡。目前尚无有效的CD治疗方法，但基因转移有可能阻止或逆转这种本来是致命的疾病的进程。我们建议使用重组腺相关病毒(AAV)载体，通过外科手术将天冬氨酸酰基酶(ASPA)基因直接注射到大脑的受影响区域。在之前的CD第一阶段研究中，使用了一种称为LPD的第一代载体，该载体由基于AAV的质粒与缩合剂(鱼精蛋白硫酸盐)和脂质体制剂(DC-Chol/DOPE)组成-外科基因转移到大脑的脑脊液空间被证明是安全的。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输送系统的不足。在过去的五年里，基因转移技术有了长足的进步，基于最近的工作，我们已经确定AAV载体作为一种基因输送系统比LPD更有效和更安全，特别是当与实质内途径结合使用时。临床前实验表明，AAV作为一种临床基因转移工具具有优越性。目前的无辅助子包装和纯化方法消除了野生型病毒重组的可能性，并提供了高纯度的载体滴度超过1012基因组颗粒/毫升。我们在啮齿动物和灵长类动物中测试的AAV载体包含一个带有额外调控后元件的哺乳动物神经启动子元件，能够驱动高水平的长期转基因表达。含有人天冬氨酸氨基转移酶基因的AAV载体(AAV-ASPA)将通过神经外科手术注射到一组Canavan病患者的大脑皮质下区域。分娩前和分娩后的评估将涉及非侵入性生化、放射、电生理、神经和认知测试。这些结果指标-NAA水平、MRI信号变化、诱发电位和标准化的神经发育评估-将被系统记录并进行统计分析。我们预计，对这项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，这项研究将作为未来所有使用人脑病毒载体进行的试验的参考。

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.