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) 是由苯丙氨酸羟化酶 (PAH) 缺乏引起的，如果不治疗，血液中苯丙氨酸 (PHE) 过多会导致神经毒性和严重发育障碍 (DD) 虽然饮食治疗可以预防 PKU 的主要临床特征，但对低水平的依从性较差。 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、多动症、白质变性、步态障碍、事实上，大多数青少年和成人的血液 PHE 浓度超出了治疗范围（≤ 360 µMol/L），此外，母体 PKU 综合征 (MPKUS) 中的宫内 PHE 暴露会导致 DD、小头畸形和心脏缺陷。需要更有效的治疗方式来提高 PHE 耐受性并减少对饮食 PHE 限制的依赖，而猪的模型则不能很好地反映人类患者的神经行为表型。生理学、解剖学和基因组与人类更相似，与啮齿类动物的无脑（光滑）大脑相比，这两种物种都具有环脑（折叠的大脑皮层），因此，缺乏 PAH 的猪应该是人类疾病的更忠实的模型。使用 CRISPR-Cas9 基因组编辑，我们在培养细胞中有效地生成了猪 PAH 外显子 6（PAHΔex6H170X 无效等位基因）的缺失和倒位，并且随后，我们生成了第一只 PKU 猪和一只杂合携带者（均为雌性），该猪分别具有两个或一个 PAH 外显子 6 的缺失等位基因，该 PKU 猪代表了典型的 PKU（血液 PHE > 1200 µMol/L），并且具有生长。为了建立 PKU 的临床前模型，我们建议建立 PAH 缺乏症（R21 相）的小型猪模型，并完全表征生化、神经发育和行为表型，以证明与人类 PKU 的等效性，并确定生物标记作为治疗测试的可靠终点（R33 阶段）。在目标 1 [R21 阶段]中，我们将培育雄性和雌性 F2 杂合猪（具有一种 PAH）。 -删除等位基因）以产生实验队列；将培育未经治疗的 PKU 雌性以产生 MPKUS 动物，临床、神经学和行为评估将识别受损的发育和行为，并进行神经病理学研究标记。在目标 2 [R33 阶段] 中，饮食治疗的 PKU（以管理神经系统表现）、饮食未治疗的 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。重点关注大脑皮层的髓鞘形成、结构和连接，以及脑白质营养不良的存在。这些和其他生物标志物（全身 PHE 氧化）将确定 PHE 之外的新结果测量。总而言之，这项研究将为 PKU 提供最佳的临床前模型，为优化治疗提供新的机会，为发现下一代脑部疾病神经疗法提供模型系统。 。