Novel gene therapy strategies for Canavan disease

描述(由申请人提供)： Canavan病(CD)是一种罕见的遗传性儿童脑白质营养不良症，由天冬氨酸氨基转移酶基因(ASPA)的常染色体隐性突变引起。虽然在广泛的族裔群体中发现了CD，但它在德系犹太人中尤其普遍，这一群体中每6 400至13 500人中就有一人受到影响。Canavan患者ASPA缺乏会导致N-乙酰-天冬氨酸(NAA)的积聚，导致脑白质肿胀和海绵状变性。这种致命性疾病的临床表现包括精神运动迟缓、低眼压、巨头畸形、脑迟缓和过早死亡。NAA由N-乙酰转移酶(NAT1)在神经元线粒体中合成，在少突胶质细胞(OLs)中被ASPA降解。中枢ASPA缺乏症的发病机制(S)以及PTS中ASPA缺乏症在Cd的病理生理学中的作用尚未得到很好的研究。目前，对CD尚无有效的临床干预措施。ASPA基因替代疗法是治疗CD的一种有吸引力的策略。早期基于第一代AAV血清2型衍生载体的基因治疗努力没有提供临床益处。这可能是由于rAAV2的转导效率不足和局部实质内载体传递的局限性所致。近年来AAV矢量学的进展产生了一些新型的重组AAVs(RAAVs)，如Kaspar等人报道的rAAV9，以及我们实验室鉴定的rAAVr.8和rAAV.10，它们在静脉注射(IV)后通过血脑屏障(BBB)高效地转导大片脑和脊髓。在这里，我们假设使用这些新的载体和给药途径，我们可以开发安全、有效和持续的基因治疗策略，纠正代谢缺陷，缓解疾病表型，延长CD小鼠的生存时间，而不会造成显著的毒性。具体地说，该项目将加深我们对CD的病理生理学，特别是在PTS中的理解，以及静脉注射后rAAV介导的CD基因治疗的机制。我们将比较野生型(Wt)和ASPA基因敲除(ASPA-/-)小鼠的血脑屏障通透性，并确定CD的最新治疗窗口。我们将对转基因盒进行优化，以更高效、更安全地表达haspA。我们将在ASPA-/-小鼠中比较我们的3个先导载体(即rAAV9、rH.8和r.10)在最小有效剂量、最新治疗窗口、长期治疗结果、免疫毒性和生物分布特征方面的差异。为了促进未来的临床发展，我们还将比较这三种载体在CD患者群体中的血清流行率，并通过过继将衣壳免疫转移到ASPA-/-小鼠来研究先前存在的免疫对中枢神经系统基因治疗的影响。这些研究将极大地促进我们目前对CD的认识，并为开发安全有效的CD患者rAAV基因治疗方法奠定基础。

DESCRIPTION (provided by applicant): Canavan disease (CD) is a rare, inherited, and fatal, childhood leukodystrophy caused by autosomal recessive mutations in the aspartoacylase gene (ASPA). Although CD has been found in a wide range of ethnic groups, it is especially prevalent in the Ashkenazi Jewish population, affecting one in 6,400 - 13,500 people in this group. ASPA deficiency in Canavan patients leads to accumulation of N-Acetyl-Aspartic Acid (NAA), resulting in swelling and spongy degeneration of white matter in the brain. The clinical manifestations of this fatal disease includ psychomotor retardation, hypotonia, macrocephaly, head lag, and early death. NAA is synthesized in the mitochondria of neurons by N-acetyltransferase (NAT1) and hydrolyzed in oligodendrocytes (OLs) by ASPA. Pathogenic mechanism(s) of ASPA deficiency in the CNS and contributions of ASPA deficits in PTs to the pathophysiology of CD are not well studied. Currently, there is no effective clinical intervention available for CD. ASPA gene replacement therapy is an attractive strategy for the treatment of CD. Earlier gene therapy efforts based on the first generation of AAV serotype 2-derived vector offered no clinical benefit. That was likely due to inadequate transduction efficiency of rAAV2 and limitations of localized intraparenchymal vector delivery. Recent advances in AAV vectorology produced some novel recombinant AAVs (rAAVs), such as rAAV9 reported by Kaspar et al., and rAAVrh.8 and rh.10 identified by our lab, that are highly efficient in transducing large areas of the brain and spinal cord by crossing the blood-brain-barrier (BBB) after intravenous (IV) injection. Here, we hypothesize that using these novel vectors and route of administration, we can develop safe, effective, and sustained gene therapy strategies that will correct the metabolic defect, alleviate the disease phenotype, and prolong survival of CD mice without causing significant toxicity. Specifically, this project will further our understanding of the pathophysiology of CD, particularly in the PTs and the mechanism of rAAV-mediated CD gene therapy after intravenous delivery. We will compare the BBB permeability between wild type (Wt) and ASPA Knockout (ASPA-/-) mice, and define the latest therapeutic window for CD. We will optimize the transgene cassette to express hASPA more efficiently and safely. We will compare our 3 lead vectors (i.e. rAAV9, rh.8, and rh.10) in ASPA-/- mice for minimum effective dose, the latest therapeutic window, long lasting therapeutic outcomes, immunotoxicity, and biodistribution profiles. To facilitate future clinical development, we will also compare these 3 vectors for sero- prevalence in the CD patient population and study impact of pre-existing immunity on CNS gene therapy by adoptively transferring capsid immunities to ASPA-/- mice. These proposed studies will significantly advance our current understanding of CD, and serve as the basis for the development of safe and effective rAAV gene therapeutics for CD patients.