Gene Transfer and NMR Studies in Alpha-Mannosidosis Brain

中枢神经系统（CNS）有效治疗的主要障碍是大多数遗传性溶酶体 储存性疾病（LSD）是所有脑细胞中的代谢缺陷导致广泛的病理学。的 大多数LSD的治疗原理是将正常酶cDNA转移到患病细胞的亚群中， 从而校正载体转导的细胞和邻近的非转导细胞， 治疗酶为了实现全局校正，转导细胞必须三维分散 因此分泌的载体编码的治疗酶可以到达所有未转导的细胞。 尽管某些AAV载体血清型的血管内注射可以穿过血脑屏障并递送给受试者，但其在体内的表达水平并不高。 由于AAV基因广泛存在于啮齿动物脑中，大型哺乳动物中的AAV载体分布主要发生在下脑中， 脊髓，限制了治疗的可能性。因此，在大型动物模型中优化载体分布 人类疾病的研究是迫切需要的，以促进转化为临床使用。 在先前的授权期，我们已经证明了一种新的AAV血清型（AAV.hu32）介导了广泛的 在由编码α-甘露糖苷酶的基因突变引起的α-甘露糖苷酶病（AMD）猫模型中的基因递送 溶酶体α-甘露糖苷酶（MANB），其重现了AMD的严重形式。值得注意的是， 编码MANB的AAV.hu32导致储存损伤的整体校正和疾病的改善。 参数虽然这似乎有希望转化为临床应用，但所需的载体剂量 将接近临床级AAV载体的生产极限， 在最近的临床试验中，使用如此高的载体剂量引起了安全问题。最后，新 初步数据显示，在早期治疗AMD猫时完全有效的载体剂量 当治疗在更晚期开始时，疾病的阶段不介导完全的脑校正。 疾病阶段，提供额外的动力，以改善全球大脑交付在较低的载体剂量。 在令人兴奋的新研究中，我们发现通过颈动脉注射比 静脉注射，表明浓缩病毒通过脑血管床 提高摄取率。令人惊讶的是，我们还发现，虽然将AAV.hu32直接注射到脑实质中， 转导的少突胶质细胞，星形胶质细胞和神经元，其血管递送导致几乎排他性的 神经元的转导。因此，目前的研究将侧重于通过确定 该载体在血液和大脑之间的路径特异性地刺激神经元， MANB产生的基因校正的神经元，并增加转导细胞的数量， 替代的给药方案将使载体在脑血管床中的摄取最大化。然后我们将 确定这些策略的优化组合是否在治疗开始时介导全局校正 在AMD猫的疾病的晚期阶段，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.