Innovation Grant to Nurture Initial Translational Efforts (IGNITE) to Neurotherapeutic Approaches in Minipig Models of PKU Disorders

苯丙酮尿症(PKU)是由苯丙氨酸羟基酶(PAH)缺乏引起的，血液中苯丙氨酸(Phe)过多会导致神经毒性和严重的发育障碍(DD)。虽然饮食治疗阻止了PKU的主要临床特征，但如果坚持低Phe饮食会导致神经发育、认知和心理问题，以及执行功能缺陷、精神症状、ADHD、脑白质变性、步态障碍和癫痫。事实上，大多数青少年和成年人超出了血型苯丙氨酸的治疗范围(≤360uMol/L)。此外，母亲PKU综合征(MPKUS)在宫内暴露Phe会导致DD、小头畸形和心脏缺陷。因此，需要更有效的治疗方法来增加对苯丙氨酸的耐受性，减少对膳食苯丙氨酸限制的依赖。PKU小鼠模型不能很好地反映人类患者的神经行为表型。相比之下，猪的生理、解剖和基因组与人类更相似，两种动物的大脑都是回脑(折叠的大脑皮层)，与啮齿类动物的无脑(光滑)大脑形成对比。因此，PAH缺乏的猪应该是更忠实的人类疾病模型。利用CRISPR-CAS9基因组编辑技术，我们在培养细胞和受精卵中高效地产生了猪PAH外显子6(PAHΔex6H170X空等位基因)的缺失和倒置。随后，我们产生了第一头PKU猪和一个杂合子携带者(均为雌性)，分别具有PAH-外显子6的两个或一个缺失等位基因。北京大学猪代表典型的北京大学(血液Phe&GT；1200微克/L)，生长迟缓和色素减退。为了建立PKU的临床前模型，我们建议建立一个PAH缺乏的小型猪模型(R21期)，并充分表征生化、神经发育和行为表型，以证明与人类PKU的等价性，并确定生物标志物作为治疗测试的可靠终点(R33期)。在目标1[R21阶段]，我们将培育雄性和雌性F2杂合子猪(带有一个PAH缺失等位基因)以产生实验队列；未处理的PKU雌性将被饲养以产生MPKUS动物。临床、神经和行为评估将确定发育和行为受损，神经病理学研究标志着R21终点。在AIM 2[R33阶段]，饲喂处理的PKU(以管理神经表现)、未处理的PKU和对照仔猪将通过神经学检查、认知和记忆测试、核磁共振和神经病理学进行神经发育表型鉴定，重点是髓鞘形成、大脑皮层的结构和连接，以及脑白质营养不良的存在。这些和其他生物标记物(全身Phe氧化)将确定Phe水平以外的新结果指标，这是FDA验证PKU治疗测试的要求。综上所述，本研究将为北京大学提供一个最佳的临床前模型，为优化治疗提供新的机会，为发现下一代脑部疾病的神经疗法提供一个模型系统。

Phenylketonuria (PKU) arises from phenylalanine hydroxylase (PAH) deficiency, with excess phenylalanine (PHE) in the blood leading to neurotoxicity and severe developmental disability (DD) if untreated. Although dietary treatment prevents major clinical features of PKU, poor adherence to a low PHE diet leads to neurodevelopmental, cognitive, and psychological problems, and deficits in executive functioning, psychiatric symptoms, ADHD, white matter degeneration, gait disturbances, and seizures. Indeed, most adolescents and adults are out of the therapeutic range for blood PHE (≤ 360 µMol/L). Additionally, in utero PHE exposure in Maternal PKU Syndrome (MPKUS) leads to DD, microcephaly, and heart defects. Thus, more effective therapeutic modalities are needed to increase PHE tolerance and reduce reliance on dietary PHE restriction. PKU mouse models poorly reflect the neurobehavioral phenotype of human patients. In contrast, pigs' physiology, anatomy, and genome are more similar to human, with both species gyrencephalic (folded cerebral cortex) in contrast to the lissencephalic (smooth) brains of rodents. Thus, a PAH-deficient pig should be a more faithful model of human disease. Using CRISPR-Cas9 genome editing, we efficiently generated deletions and inversions of porcine PAH exon 6 (PAHΔex6H170X null alleles) in cultured cells and zygotes. Subsequently, we generated the first PKU pig and a heterozygous carrier (both females) having two or one deletion alleles of PAH-exon 6, respectively. The PKU pig represents classic PKU (blood PHE >1200 µMol/L), with growth retardation and hypopigmentation. To establish a pre-clinical model for PKU, we propose to generate a minipig model of PAH deficiency (R21 phase) and to fully characterize biochemical, neurodevelopmental and behavioral phenotypes to demonstrate equivalence to human PKU and to identify biological markers as reliable endpoints for therapeutic testing (R33 phase). In Aim 1 [R21 phase], we will breed male and female F2 heterozygous pigs (with one PAH-deletion allele) to generate experimental cohorts; untreated PKU females will be bred to generate MPKUS animals. Clinical, neurological and behavioral evaluation will identify compromised development and behavior, with neuropathological studies marking the R21 endpoint. In Aim 2 [R33 phase], dietary-treated PKU (to manage neurologic presentation), dietary-untreated PKU, and control piglets will undergo neurodevelopmental phenotyping by neurological exam, cognitive and memory testing, MRI, and neuropathology, with a focus on myelination, structure and connections of the cerebral cortex, and presence of leukodystrophy. These and other biological markers (whole body PHE oxidation) will identify new outcome measures beyond PHE levels, as required by the FDA for verifying therapeutic testing in PKU. In summary, this study will provide an optimal pre-clinical model for PKU to allow new opportunities to optimize therapy, providing a model system for discovery of next generation neurotherapeutics of brain disorders.