Novel gene therapy strategies for Canavan disease

描述（由申请人提供）： Canavan病（CD）是一种罕见的、遗传性的、致命的儿童脑白质营养不良，由乙酰化酶基因（ASPA）的常染色体隐性突变引起。虽然CD已在广泛的种族群体中发现，但它在德系犹太人中尤其普遍，影响该群体中每6，400 - 13，500人中的一人。Canavan患者的ASPA缺乏导致N-乙酰天冬氨酸（NAA）蓄积，导致脑中白色物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发育迟滞、肌张力减退、大头畸形、头滞后和早期死亡。NAA由N-乙酰转移酶（NAT 1）在神经元线粒体中合成，并由ASPA在少突胶质细胞（OLs）中水解。CNS中ASPA缺乏的致病机制以及PT中ASPA缺乏对CD病理生理学的贡献尚未得到充分研究。目前，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。基于第一代AAV血清型2衍生载体的早期基因治疗努力没有提供临床益处。这可能是由于rAAV 2的转导效率不足和局部脑实质内载体递送的局限性。AAV载体学的最新进展产生了一些新的重组AAV（rAAV），例如Kaspar等人报道的rAAV 9，以及我们实验室鉴定的rAAVrh.8和rh.10，其在静脉内（IV）注射后通过穿过血脑屏障（BBB）在大面积脑和脊髓中高效转导。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，持续的基因治疗策略，将纠正代谢缺陷，减轻疾病表型，并延长生存的CD小鼠，而不会引起显着的毒性。具体而言，该项目将进一步加深我们对CD的病理生理学的理解，特别是在PT和静脉内递送后rAAV介导的CD基因治疗的机制中。我们将比较野生型（Wt）和ASPA敲除（ASPA-/-）小鼠之间的BBB通透性，并确定CD的最新治疗窗口。我们将优化转基因盒以更有效和安全地表达hASPA。我们将在ASPA-/-小鼠中比较我们的3种先导载体（即rAAV 9、rh.8和rh.10）的最小有效剂量、最新治疗窗口、持久治疗结果、免疫毒性和生物分布特征。为了促进未来的临床开发，我们还将比较这3种载体在CD患者人群中的血清流行率，并通过将衣壳免疫过继转移至ASPA-/-小鼠来研究既存免疫对CNS基因治疗的影响。这些拟议的研究将显着推进我们目前对CD的理解，并作为CD患者的安全和有效的rAAV基因疗法的发展的基础。

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.