Improving AAV-based gene therapy for Krabbe Disease

摘要 Krabbe病（KD）是一种罕见的遗传性脑白质营养不良引起的GALC基因突变 编码溶酶体酶半乳糖神经酰胺酶（GALC）。缺乏功能性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， 退化，无法茁壮成长，最终在2-3岁时死亡。川崎病治疗造血干细胞 细胞移植（HSCT），它减缓了疾病的进展，延长了预期寿命， 青少年时期，但它不是一种治疗方法。腺相关病毒（Adeno-associated viruses，AAV）已成功地用作基因工程， 治疗载体在许多临床前和临床试验。腺相关病毒基因治疗在癌症中的初步成功 主要影响神经元细胞的疾病导致了对它们在单基因疾病中的功效的研究， 多种细胞类型，如KD。Bongarzone实验室先前开发了一种使用AAV 9的治疗方案， GALC以纠正KD的Twitch鼠模型中的缺陷。这种治疗完全防止了 在生命的前6-8个月的疾病发展，使得AAV处理的小鼠几乎无法区分 从野生型小鼠身上。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，包括小鼠中疾病体征和局灶性脱髓鞘斑块的发展， 高龄;其他研究人员也注意到了类似的发现。无替代修改 迄今为止已经提出了改进基因疗法。 本申请的首要目标是确定基因治疗疗效是否由于以下原因而下降： 成年人大脑中AAV DNA的耗竭以及如何优化基因治疗的疗效和持续时间， 抽搐的老鼠。由于AAV-GALC DNA在进入染色体后作为非复制的染色体外附加体存在， 细胞，我们假设治疗功效的下降是由复制细胞中治疗载体的丢失引起的， 特别是少突胶质细胞前体，在Twitcher脑中，这导致平均AAV- GALC游离体/细胞随时间的变化。这一假设将通过在小鼠出生后不久用 AAV 9-GALC，然后基于临床表现观察治疗有效多长时间。 评分、存活率以及生物化学和组织学分析。然后我们将检查是否重新给药利用 少突胶质细胞靶向的AAV（AAV-001）在生命后期，但在疾病体征发作之前，延迟或预防 疾病迹象的发展。最后，我们将确定单独使用AAV-001是否会增加疗效。 和治疗持续时间。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， 治疗KD患者。此外，我们的研究结果将产生内在的影响，更好地描述附加体如何 AAV DNA在类似的单基因疾病中表现。

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.