Gene Transfer and NMR Studies in Alpha-Mannosidosis Brain

大多数遗传性溶酶体患者有效治疗中枢神经系统(CNS)的主要障碍 贮藏性疾病(LSD)是指所有脑细胞的代谢缺陷导致广泛的病理改变。这个 大多数LSD的治疗原理是将正常的酶cDNA转移到疾病细胞的子集中， 从而纠正载体转导的细胞和邻近的非转导细胞吸收分泌的 治疗性酵素。为了实现全局校正，换能器单元必须以三维方式分散 这样分泌的载体编码的治疗酶可以到达所有未转导的细胞。 尽管血管内注射某些AAV载体血清型可以跨越血脑屏障并传递 基因广泛存在于啮齿动物的大脑中，AAV载体在大型哺乳动物中的分布主要发生在下脑和 脊髓，限制了治疗的潜力。因此，在大型动物模型中优化媒介分布 为了促进将其转化为临床应用，迫切需要对人类疾病进行研究。 在之前的资助期间，我们已经证明了一种新的AAV血清型(AAV.hu32)介导了广泛的 编码基因突变导致的α-甘露糖沉着症(AMD)猫模型的基因传递 溶酶体a-甘露糖苷酶(MANB)，它概括了AMD的严重形式。值得注意的是，大量的 编码MANB的AAV.hu32导致存储损伤的全局纠正和疾病的改善 参数。尽管这看起来很有希望转化为临床使用，但所需的媒介剂量 当扩大到人类患者时，将接近临床级AAV载体的生产极限 在最近的临床试验中，人们对使用如此高的媒介剂量提出了安全担忧。最后，新的 初步数据显示，当AMD猫在早期治疗时完全有效的媒介剂量 当在更晚期开始治疗时，疾病的各个阶段并不能调解完全的大脑矫正。 疾病阶段，提供额外的动力，以较低的媒介剂量改善全球脑递送。 在令人兴奋的新研究中，我们发现通过颈动脉注射比通过颈动脉注射更有效 静脉注射，表明浓缩的病毒通过大脑的血管床 提高摄取率。令人惊讶的是，我们还发现，当将AAV.hu32直接注射到脑实质中时 转导少突胶质细胞、星形胶质细胞和神经元，其血管输送导致几乎排他 神经元的转导。因此，目前的研究将集中于通过确定以下方法来减少所需的媒介剂量 载体通过血液和大脑之间的途径来特异性地转导神经元，增加了 MANB由遗传校正的神经元产生，并通过增加转导细胞的数量 另一种剂量方案，将最大限度地提高脑血管床中的媒介摄取。到时候我们会的 确定这些策略的优化组合是否在治疗开始时调解全局纠正 在AMD猫的疾病晚期，这将是大多数人类患者的情况。

A major barrier to effective treatment of the central nervous system (CNS) in most inherited lysosomal storage diseases (LSD) is that the metabolic defect in all brain cells results in widespread pathology. The therapeutic principle for most LSDs is to transfer a normal enzyme cDNA into a subset of diseased cells, thereby correcting both vector-transduced cells and neighboring non-transduced cells that take up the secreted therapeutic enzyme. To achieve global correction, transduced cells must be dispersed 3-dimensionally throughout the brain so that secreted vector-encoded therapeutic enzyme can reach all non-transduced cells. Although intravascular injection of certain AAV vector serotypes can cross the blood-brain barrier and deliver genes widely in rodent brains, AAV vector distribution in large mammals occurs mostly in the lower brain and spinal cord, limiting the potential for treatment. Thus, optimization of vector distribution in large animal models of human diseases is critically needed to facilitate translation into clinical usage. In the prior grant period, we have shown that a novel AAV serotype (AAV.hu32) mediates widespread gene delivery in a cat model of alpha-mannosidosis (AMD) caused by a mutation in the gene encoding lysosomal a-mannosidase (MANB) that recapitulates the severe form of AMD. Notably, high doses of AAV.hu32 encoding MANB resulted in global correction of the storage lesions and improvements in disease parameters. Although this appears promising for translation into clinical usage, the vector doses required would be near the limits of production for clinical grade AAV vector when scaled up to human patients and there have been safety concerns raised in recent clinical trials for use of such high vector doses. Finally, new preliminary data shows that vector doses that are completely effective when AMD cats are treated in the early stages of the disease do not mediate complete brain correction when treatment is initiated at a more advanced stage of disease, providing additional impetus to improve global brain delivery at lower vector doses. In exciting new studies, we have found that injection via carotid artery was even more effective than intravenous injection, suggesting that passage of concentrated virus through the vascular bed of the brain improves uptake. Surprisingly, we also found that while direct injection of AAV.hu32 into brain parenchyma transduced oligodendrocytes, astrocytes and neurons, its vascular delivery resulted in the almost exclusive transduction of neurons. Thus, current studies will focus on reducing the required vector dose by determining the route the vector takes between blood and brain to specifically transduce neurons, increasing the levels of MANB produced by the genetically corrected neurons, and increasing the number of transduced cells by an alternative dosing regimen that will maximize vector uptake in the vascular bed of the brain. We will then determine if an optimized combination of these strategies mediates global correction when treatment is started at the advanced stage of disease of AMD cats, which will be the case for most human patients.