Gene Therapy Clinical Candidate Development for Carbamoyl Phosphate Synthetase Deficiency

氨基甲酰磷酸合成酶缺乏症的基因治疗临床候选药物开发

• 批准号: 10339836
• 负责人: Gerald S Lipshutz
• 金额: $ 38.92万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10339836
• 关键词: Address; Affect; Ammonia; Animal Model; Anorexia; Biotechnology; Birth; Brain; Carbamyl Phosphate; Cerebral Edema; Clinical; Clinical Protocols; Codon Nucleotides; Coma; Complementary DNA; Cryopreservation; DNA cassette; Data; Defect; Dependovirus; Development; Diet; Disease; Disease model; Dose; Drug Metabolic Detoxication; Encephalopathies; Enhancers; Enzymatic Biochemistry; Enzymes; Gene Delivery; Gene Transduction Agent; Genes; Genetic; Genetic Diseases; Genome; Glutamine; Goals; Hepatic; Hepatocyte; Hereditary Disease; Human; Hybrids; Hyperammonemia; Hyperargininemia; Hypoventilation; Impairment; Incidence; Infant; Knockout Mice; Laboratories; Lethargies; Life; Ligase; Liver; Mammals; Mediating; Medical; Mendelian disorder; Metabolic Pathway; Mission; Mitochondrial Proteins; Morbidity - disease rate; Mus; Mutation; N acetyl L glutamate; Neonatal; Neonatal Mortality; Nervous System Trauma; Neurologic; Nitrogen; Organ; Pathway interactions; Patients; Periodicity; Phase; Phenotype; Plasma; Posture; Pre-Clinical Model; Prevention strategy; Production; Proteins; Public Health; Publishing; Regulator Genes; Regulatory Element; Research; Risk; Seizures; Serotyping; Site; Survivors; Technology; Testing; Therapeutic; Transgenes; Transgenic Mice; Transplantation; United States National Institutes of Health; Urea cycle disorders; Vectorial capacity; Viral Vector; Vulnerable Populations; adeno-associated viral vector; base; clinical application; clinical candidate; clinical development; clinically translatable; cost; critical period; disability; effective therapy; enzyme deficiency; experience; gene product; gene replacement; gene therapy; genetic approach; high risk; homologous recombination; human disease; humanized mouse; immunosuppressed; in vivo; liver transplantation; liver xenograft; model development; mortality; mortality risk; mouse model; natural hypothermia; neonate; novel; novel therapeutic intervention; novel therapeutics; postnatal development; pre-clinical; pre-clinical therapy; programs; promoter; severe intellectual disability; treatment strategy; urea cycle; vector; wasting

Project Summary/Abstract The urea cycle is the major pathway for detoxification of ammonia in mammals. Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a neurologically-devastating condition that, while late- onset does occur, the disorder primarily is a condition affecting neonates. The deficiency of this enzyme is characterized clinically by periodic episodes of hyperammonemia resulting in progressive irreversible neurological injury and severe CNS impairment, particularly during a period of critical postnatal development; the condition is associated with a high likelihood of early neonatal mortality. The long-term goal of this program is to develop a clinical candidate gene therapy vector that will result in ammonia control and restore ureagenesis in CPS1 deficiency. Recent advances in gene therapy have led to the concept of using adeno-associated virus (AAV)-based biotechnology to treat CPS1 deficiency; at present, this monogenic disorder has no completely effective therapy except liver transplantation, though often not occurring until the infant has reached a size where successful transplantation is more likely but multiple episodes of neurological injury have been sustained. In addition to being one of the most difficult urea cycle disorders to treat clinically, the development of a gene therapy approach has been hampered by several challenges. First, until recently, there were no animal models of the disorder. Second, CPS1, the most abundant mitochondrial protein in the human liver, must be expressed at high level in hepatocytes. And third, the size of the CPS1 cDNA at 4.5kb places limitations on the expression cassette size; this may be the greatest hindrance for a clinically translatable vector that can be produced at high titer. This proposal seeks to advance a gene therapy approach using small hepatocyte-specific gene regulatory elements to produce a compact CPS1-expressing AAV and a hybrid/dual vector AAV as approaches for this poorly treated disorder. The Lipshutz laboratory recently developed two murine models of CPS1 deficiency and has published and preliminary data on the efficacy of an AAV approach; this proposal is to complete the development and bring forth a clinical candidate vector. Preliminary and published data: The research group has: 1) developed a conditional Cps1-deficient knockout mouse, replicating neonatal onset CPS1 deficiency; 2) demonstrated hepatocyte-specific expression of CPS1 from single vector AAV that has led to plasma ammonia control; 3) developed a dual (or split) AAV approach to express CPS1 in the liver; and 4) has produced humanized mice where CPS1-deficient hepatocytes have repopulated the murine liver; the latter is essential for testing these vector approaches. Specific Aim 1: Optimize the AAV constructs for hepatic CPS1 expression and identify the optimal candidate to advance. Specific Aim 2: Test different hepatotropic serotypes of AAV vector- based CPS1 expression utilizing the optimized transgene cassette in a Cps1-deficient humanized mouse model. The proposed research is significant as it is expected that at completion a final clinical candidate and serotype will have been chosen for establishing hepatic gene replacement for CPS1 deficiency.

项目摘要/摘要尿素循环是哺乳动物氨解毒的主要途径。 氨基甲酰磷酸合成酶 1 (CPS1) 缺乏症是一种神经系统破坏性病症，虽然晚期 如果确实发生了这种疾病，那么这种疾病主要是影响新生儿的病症。这种酶的缺陷是 临床特征是周期性发作的高氨血症导致进行性不可逆 神经损伤和严重的中枢神经系统损伤，特别是在产后发育的关键时期； 这种情况与新生儿早期死亡的可能性很高有关。该计划的长期目标 是开发一种临床候选基因治疗载体，该载体将导致氨控制并恢复尿素生成 CPS1 缺乏症。基因治疗的最新进展引发了使用腺相关病毒的概念 基于 AAV 的生物技术治疗 CPS1 缺陷；目前，这种单基因疾病还没有完全 除肝移植外的有效疗法，但通常要等到婴儿达到一定尺寸后才进行 移植成功的可能性更大，但神经损伤已持续多次。 除了是临床上最难治疗的尿素循环障碍之一之外，基因的发展 治疗方法受到一些挑战的阻碍。首先，直到最近，还没有动物模型 的疾病。其次，必须表达人类肝脏中最丰富的线粒体蛋白CPS1 在肝细胞中呈高水平。第三，CPS1 cDNA 的大小为 4.5kb，这限制了表达 盒式磁带尺寸；这可能是可在高水平生产的临床可翻译载体的最大障碍 效价。该提案旨在利用小肝细胞特异性基因调控来推进基因治疗方法 产生紧凑的 CPS1 表达 AAV 和混合/双载体 AAV 作为实现此目的的方法 治疗不当的疾病。 Lipshutz 实验室最近开发了两种 CPS1 缺陷小鼠模型 已发布有关 AAV 方法功效的初步数据；该提案旨在完成 开发并提出临床候选载体。初步和已发表的数据：研究小组 已：1) 开发了条件性 Cps1 缺陷敲除小鼠，复制了新生儿发病的 CPS1 缺陷； 2） 证明单载体 AAV 的 CPS1 肝细胞特异性表达可导致血浆氨 控制; 3）开发了双重（或分离）AAV方法在肝脏中表达CPS1； 4) 已产生 人源化小鼠，其中 CPS1 缺陷的肝细胞已重新填充到小鼠肝脏中；后者对于 测试这些矢量方法。具体目标 1：优化 AAV 构建体以实现肝脏 CPS1 表达和 确定晋升的最佳候选人。具体目标2：测试AAV载体的不同亲肝血清型- 在 Cps1 缺陷的人源化小鼠模型中利用优化的转基因盒进行基于 CPS1 的表达。 拟议的研究意义重大，因为预计完成后将得出最终的临床候选者和血清型 将被选择用于建立针对 CPS1 缺陷的肝基因替代。