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

创新资助培育小型猪 PKU 疾病模型神经治疗方法的初步转化努力 (IGNITE)

• 批准号: 9372728
• 负责人: Steven F Dobrowolski
• 金额: $ 38.36万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9372728
• 关键词: Adherence; Adolescent; Adult; Affect; Age; Alleles; Anatomy; Animals; Attention deficit hyperactivity disorder; Behavior; Behavioral; Biochemical; Biochemistry; Biological Markers; Biological Models; Blood; Body Size; Brain; Brain Diseases; CRISPR/Cas technology; Cerebral cortex; Childhood; Classical phenylketonuria; Clinical; Clinical Treatment; Clustered Regularly Interspaced Short Palindromic Repeats; Cognitive; Cultured Cells; Defect; Development; Developmental Disabilities; Diet; Disease; Early identification; Embryopathy; Evaluation; Exhibits; Exons; Family suidae; Feeding behaviors; Female; Frequencies; Functional disorder; Gait abnormality; Generations; Genes; Genome; Grant; Growth; Growth and Development function; Guide RNA; Heart Abnormalities; Hippocampus (Brain); Human; Hypopigmentation; Impairment; Individual; Intake; Intervention; Learning; Magnetic Resonance Imaging; Maternal Phenylketonuria; Measures; Memory; Metabolic; Microcephaly; Miniature Swine; Modality; Modeling; Mothers; Motor Activity; Mus; Neonatal; Neonatal Screening; Nervous System Physiology; Neurocognitive; Neurologic; Outcome; Outcome Measure; Patients; Phase; Phenotype; Phenylalanine; Phenylalanine Hydroxylase; Phenylketonurias; Physiology; Pre-Clinical Model; Prefrontal Cortex; Radial; Rodent; Rodent Model; Secondary to; Seizures; Serum; Spinal Cord; Structure; Syndrome; Testing; Therapeutic; Toxic effect; Translations; Treatment Efficacy; arm; brain size; clinical phenotype; cohort; dietary restriction; executive function; experience; genome editing; human disease; improved; in utero; innovation; leukodystrophy; lissencephaly; male; mouse model; myelination; neurobehavioral; neurodevelopment; neuropathology; neuropsychiatry; neurotoxicity; next generation; novel therapeutics; object recognition; oxidation; pre-clinical; premature; prevent; psychiatric symptom; psychologic; structural heart disease; therapeutic evaluation; white matter; zygote

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.

苯丙酮尿症（PKU）由苯丙氨酸羟化酶（PAH）缺乏引起，血液中过量的苯丙氨酸（PHE）如果不治疗会导致神经毒性和严重的发育障碍（DD）。虽然饮食治疗可以预防PKU的主要临床特征，但对低PHE饮食的依从性差会导致神经发育、认知和心理问题，以及执行功能缺陷、精神症状、ADHD、白色变性、步态障碍和癫痫发作。事实上，大多数青少年和成人的血PHE超出了治疗范围（≤ 360 µMol/L）。此外，母体PKU综合征（MPKUS）的子宫内PHE暴露导致DD、小头畸形和心脏缺陷。因此，需要更有效的治疗方式来增加PHE耐受性并减少对饮食PHE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，猪的生理学、解剖学和基因组与人类更相似，两个物种的脑回（折叠的大脑皮层）与啮齿动物的无脑（光滑）大脑形成对比。因此，PAH缺陷猪应该是人类疾病的更可靠模型。使用CRISPR-Cas9基因组编辑，我们在培养的细胞和受精卵中有效地产生了猪PAH外显子6（PAHΔ ex 6 H170 X无效等位基因）的缺失和倒位。随后，我们产生了第一个PKU猪和杂合子携带者（均为女性），分别具有两个或一个PAH外显子6的缺失等位基因。PKU猪代表典型的PKU（血液PHE >1200 µMol/L），伴有生长迟缓和色素减退。为了建立PKU的临床前模型，我们建议生成PAH缺乏症的小型猪模型（R21阶段），并充分表征生化、神经发育和行为表型，以证明与人PKU的等效性，并确定生物标志物作为治疗试验的可靠终点（R33阶段）。在目标1 [R21阶段]，我们将饲养雄性和雌性F2杂合猪（具有一个PAH缺失等位基因）以生成实验队列;将饲养未处理的PKU雌性以生成MPKUS动物。临床、神经学和行为学评估将确定受损的发育和行为，神经病理学研究标记R21终点。在目标2 [R33阶段]，饮食处理的PKU（以管理神经表现）、饮食未处理的PKU和对照仔猪将通过神经系统检查、认知和记忆测试、MRI和神经病理学进行神经发育表型分析，重点关注髓鞘形成、大脑皮质的结构和连接以及是否存在脑白质营养不良。这些和其他生物标志物（全身PHE氧化）将识别超出PHE水平的新结局指标，如FDA要求的那样，用于验证PKU的治疗试验。总之，这项研究将为PKU提供一个最佳的临床前模型，为优化治疗提供新的机会，为发现下一代脑疾病的神经治疗提供一个模型系统。