Improving AAV-based gene therapy for Krabbe Disease

摘要 Krabbe病是一种罕见的遗传性脑白质营养不良，由GALC基因突变引起 编码溶酶体酶半乳糖神经酰胺酶(GALC)。缺乏有效的GALC导致全球 脱髓鞘和神经变性。未经治疗的KD在婴儿中表现为发育迟缓和 退化，无法茁壮成长，最终在2-3岁时死亡。用造血干细胞治疗川崎病 细胞移植(HSCT)可以减缓疾病的进展，延长人类的预期寿命。 十几岁，但这不是治愈的方法。腺相关病毒(AAV)已被成功地作为基因利用 大量临床前和临床试验中的治疗治疗载体。甲型肝炎病毒基因治疗的初步成功 主要影响神经细胞的疾病导致了对影响神经细胞的单基因疾病疗效的调查 多种细胞类型，如KD。邦加宗实验室此前开发了一种使用AAV9的治疗方案- GALC纠正抽动小鼠KD模型的不足。这种治疗完全预防了 在生命的前6-8个月，疾病的发展，以至于用AAV治疗的小鼠几乎无法区分 来自野生型小鼠。尽管有这些有希望的结果，我们的研究也揭示了治疗的缓慢下降 随着时间的推移，疗效，包括疾病体征和局灶性脱髓鞘斑块的发展 高龄；其他调查人员也注意到了类似的发现。没有替代的修改 到目前为止，人们已经提出了改进基因治疗的建议。 这项应用的首要目标是确定基因治疗的有效性是否由于 成人脑内AAV DNA的耗竭以及如何优化基因治疗的疗效和持续时间 抽动小鼠。由于AAV-GALC DNA在进入AAV-GALC后以非复制性的染色体外集落形式存在 细胞，我们假设治疗效果的下降是由于复制细胞中治疗载体的丢失所致， 特别是少突胶质细胞前体细胞在抽动者脑中，这导致平均AAV- 随着时间的推移，每个细胞的GALC内切体。这一假说将通过在小鼠出生后不久用 AAV9-GALC减量给药，根据临床疗效观察疗效 评分、存活率以及生化和组织学分析。然后我们将检查是否重新使用 少突胶质细胞靶向AAV(AAV-001)的时间较晚，但在疾病症状出现之前，延迟或预防 疾病体征的发展。最后，我们将确定单独使用AAV-001是否会增加疗效 和疗程与AAV9比较。这项研究很重要，因为我们需要更好地 在尝试使用基于AAV的疗法之前，了解外周AAV DNA发生了什么 治疗KD患者。此外，我们的发现将产生内在的影响，更好地刻画上体如何 AAV DNA在类似的单基因疾病中表现出来。

Abstract Krabbe’s Disease (KD) is a rare inherited leukodystrophy caused by mutations in the GALC gene encoding the lysosomal enzyme, galactosylceramidase (GALC). Lack of functional GALC leads to global demyelination and neurodegeneration. Untreated KD presents in infants as developmental delay and regression, failure to thrive, and ultimately death by 2-3 years of age. KD is treated with hematopoietic stem cell transplantation (HSCT) which slows the progression of disease and prolongs life expectancy into the teenage years, but it is not a cure. Adeno-associated viruses (AAV) have been utilized successfully as gene therapy treatment vectors in numerous pre-clinical and clinical trials. The initial success of AAV gene therapy in diseases primarily affecting neuronal cells led to investigations of their efficacy in monogenic diseases affecting multiple cells types such as KD. The Bongarzone lab previously developed a treatment protocol using AAV9- GALC to correct the deficiency in the Twitcher murine model of KD. This treatment completely prevented disease development for the first 6-8 months of life, such that AAV-treated mice were nearly indistinguishable from wild-type mice. Despite these promising results, our study also revealed a slow decline in treatment efficacy over time, including development of disease signs and focal demyelinating plaques in mice of advanced age; similar findings have been noted by other investigators as well. No alternative modifications to improve gene therapy have been proposed thus far. The overarching goal of this application is to establish whether gene therapy efficacy is declining due to exhaustion of AAV DNA in the adult brain and how to optimize gene therapy treatment efficacy and duration in Twitcher mice. As AAV-GALC DNA exists as a non-replicating extra-chromosomal episome after entering a cell, we hypothesize the decline in treatment efficacy is caused by loss of therapeutic vector in replicating cells, particularly oligodendrocyte precursors, in the Twitcher brain, which leads to a decrease in the average AAV- GALC episomes per cell over time. This hypothesis will be investigated by treating mice shortly after birth with AAV9-GALC at decreasing dosages and then observing how long the treatment is efficacious based on clinical score, survival, as well as biochemical and histological analyses. We will then examine if redosing utilizing oligodendrocyte targeted AAV (AAV-001) later in life, but prior to disease sign onset, delays or prevents development of disease signs. Finally, we will determine if utilizing AAV-001 singly will increase the efficacy and duration of treatment in comparison to AAV9. This study is important because we need to better understand what happens to episomal AAV DNA long term before attempting to use AAV based therapies to treat KD patients. Furthermore, our findings will have the intrinsic impact of better characterizing how episomal AAV DNA behaves in similar monogenic disorders.