Molecular Genetics Of Heritable Human Disorders

人类遗传性疾病的分子遗传学

• 批准号: 10685188
• 负责人: JANICE CHOU
• 金额: $ 160.25万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10685188
• 关键词: 5' -AMP-activated protein kinase; Active Sites; Acute Renal Failure with Renal Papillary Necrosis; Adenine; Age; Albumins; Alleles; Angiotensinogen; Animal Model; Attenuated; Autophagocytosis; Autophagosome; Blood; Blood Glucose; CRISPR therapeutics; CRISPR/Cas technology; Carcinoma; Caucasians; Chronic Kidney Failure; Clinical; Code; Codon Nucleotides; Collaborations; Complex; Cooperative Research and Development Agreement; Couples; Cytoplasm; Data; Development; Diet therapy; Disease; Disease model; Down-Regulation; Endoplasmic Reticulum; Enhancers; Etiology; Excretory function; Exons; Extracellular Matrix Proteins; Face; Family member; Fasting; Fatty Liver; Fibrosis; Functional disorder; Future; GTP-Binding Protein alpha Subunits, Gs; Gene Transfer; Generations; Genes; Genome; Genomic DNA; Glucose-6-Phosphate; Glucosephosphates; Glycogen; Glycogen Storage Disease Type I; Goals; Growth; Guide RNA; Hepatic; Hepatocarcinogenesis; Hepatomegaly; Heritability; Hour; Human; Hydrolysis; Hyperlipidemia; Hyperuricemia; Hypoglycemia; Impairment; Initiator Codon; Inorganic Phosphate Transporter; Integral Membrane Protein; Interphase Cell; Intestines; Kidney; Kidney Diseases; Kidney Failure; Laboratories; Lead; Legal patent; Link; Liver; Liver Cell Adenoma; Mediating; Messenger RNA; Metabolic; Molecular Genetics; Mouse Strains; Mus; Mutation; Newborn Infant; Nonsense Codon; Organelles; Outcome; Pathogenicity; Pathologic Processes; Pathway interactions; Patients; Pharmacologic Substance; Phase I/II Clinical Trial; Phase III Clinical Trials; Phosphorylation; Phosphotransferases; Process; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Recombinant adeno-associated virus (rAAV); Renin; Renin-Angiotensin System; Reporting; Risk; SIRT1 gene; Seizures; Signal Transduction; Smooth Muscle Actin Staining Method; System; Technology; Therapeutic; Time; Transcript; Triglycerides; Untranslated Regions; Up-Regulation; Variant; Work; adeno-associated viral vector; alpha catenin; base; base editing; base editor; blood glucose regulation; efficacy evaluation; environmental stressor; gene therapy; genetic technology; genomic locus; glucose production; glucose-6-phosphatase; hypercalciuria; in vivo; inhibitor; insertion/deletion mutation; kidney fibrosis; lipid nanoparticle; metabolic phenotype; novel therapeutics; nutrient deprivation; overexpression; postnatal development; programs; promoter; therapeutically effective; transcription factor; urinary; vector; virtual

The rAAV-co-G6PC vector used in the current phase I/II clinical trial is episomally expressed and the long-term durability of expression in humans is currently being established. We therefore sought to explore the use of the CRISPR/Cas9 technology to correct a pathogenic GSD-Ia variant in its native genetic locus. The most prevalent pathogenic mutation identified in Caucasian GSD-Ia patients is G6PC-p.R83C, representing 32% of diseased alleles. Using the CRISPR/Cas9-based gene editing technology, we generated a GSD-Ia mouse disease model, the G6pc-R83C mouse homozygous for the G6PC-p.R83C mutation and showed that the G6pc-R83C mice manifest impaired glucose homeostasis mimicking that of human GSD-Ia. We then used a CRISPR/Cas9-based gene editing system to treat newborn G6pc-R83C mice and showed that the treated mice grew normally to age 16 weeks without hypoglycemia seizures. The treated G6pc-R83C mice, expressing 3% of normal hepatic G6Pase- activity, maintained glucose homeostasis, displayed normalized blood metabolites, and could sustain 24 hours of fasting. Taken together, we have developed a second-generation therapy in which in vivo correction of a pathogenic G6PC-p.R83C variant in its native genetic locus could lead to potentially permanent, durable, long-term correction of the GSD-Ia disorder. Clinically, GSD-Ib patients manifest a metabolic phenotype of impaired blood glucose homeostasis and long-term risk of hepatocellular adenoma/carcinoma (HCA/HCC). The etiology of HCA/HCC in GSD-Ib is unknown. Studies have shown that deficiency in autophagy, an evolutionary conserved, degradative process that produces energy and building blocks through lysosomal degradation of intracellular proteins and organelles in times of nutrient deprivation and environmental stresses, contributes to hepatocarcinogenesis. Autophagy can be regulated positively by sirtuin 1 (SIRT1), AMP-activated protein kinase (AMPK), and forkhead box O (FoxO) transcription factor family members. In the liver, AMPK is activated via phosphorylation of the AMPK -subunit at residue T172 by the liver kinase B-1 (LKB1), a serine/threonine kinase. To understand the pathways contributing to hepatocarcinogenesis in GSD-Ib, we hypothesized that impaired hepatic autophagy is a significant contributor. In this study, we show that G6PT deficiency leads to impaired hepatic autophagy evident from attenuated expression of many components of the autophagy network, decreased autophagosome formation, and reduced autophagy flux. The G6PT-deficient liver displayed impaired SIRT1 and AMPK signaling, along with reduced expression of SIRT1, FoxO3a, LKB1, and the active p-AMPK. Importantly, we show that overexpression of either SIRT1 or LKB1 in G6PT-deficient liver restored autophagy and SIRT1/FoxO3a and LKB1/AMPK signaling. The hepatosteatosis in G6PT-deficient liver decreased SIRT1 expression. LKB1 overexpression reduced hepatic triglycerides levels, providing a potential link between LKB1/AMPK signaling upregulation and the increase in SIRT1 expression. In conclusion, downregulation of SIRT1/FoxO3a and LKB1/AMPK signaling underlies impaired hepatic autophagy which may contribute to HCA/HCC development in GSD-Ib. Understanding this mechanism may guide future therapies. We have generated 4 efficacious G6PC gene transfer rAAV vectors for GSD-Ia gene therapy; rAAV-G6PC expressing the wild-type (WT) G6PC, rAAV-coG6PC expressing a codon-optimized (co) G6PC, rAAV-G6PC-S298C expressing a G6PC-S298C variant with increased efficacy, and rAAV-coG6PC-S298C. Our rAAV-G6PC/rAAV-coG6PC vector (US patent #9,644,216) technology was licensed to Ultragenyx Pharmaceutical Inc who has launched a phase I/II clinical trial (NCT03517085) in 2018 and followed by a phase III clinical trial (NCT05139316) in 2022 using the rAAV-GPE-coG6PC vector. To examine the long-term efficacy of these rAAV vectors, we conducted a long-term (66-76 week) gene transfer study in G6pc-/- mice using these rAAV vectors. All treated G6pc-/- mice survived to age 66-76 weeks, and the outcomes were additive. Hepatic G6Pase- activities in rAAV-G6PC-S298C-, rAAV-coG6PC-, and rAAV-coG6PC-S298C-treated G6pc-/- mice were 1.7-, 1.7-, and 4.4-fold higher, respectively than that in rAAV-G6PC-WT-treated mice. The efficacy of the rAAV-coG6PC-S298C vector is 2.6-fold higher than the rAAV-coG6PC vector currently used in phase III clinical trial (NCT05139316). Taken together, the rAAV-G6PC-S298C and rAAV-coG6PC-S298C vectors offer attractive clinical alternatives. We explore the Adenine base editor (ABE)-based technologies that enable a programmable conversion of AT to GC in genomic DNA for GSD-Ia therapies. The ABE system works in both dividing and non-dividing cells, is reported to produce virtually no indels or off-target editing in the genome, can correct a pathogenic variant in its native genetic locus, leading to permanent, therapeutically effective long-term expression. This is a collaborative study with Beam Therapeutics, Cambridge, MA under a CRADA. The G6PC-p.R83C is the most prevalent pathogenic mutation identified in Caucasian GSD-Ia patients that contains a single G>A transition in the G6PC gene. We first generated a homozygous humanized R83C/R83C mouse strain, the G6PC-R83C mouse by inserting the entire coding sequence of the human G6PC-p.R83C along with human G6PC 3-UTR into exon 1 of the mouse G6pc gene at the ATG start codon. This insertion places the human transcript under the control of the native mouse G6pc promoter/enhancer. The mouse G6pc gene is disrupted by a premature STOP codon created in the mouse G6pc exon 1. We showed that the G6PC-R83C mice manifest impaired glucose homeostasis characterized by growth retardation, hypoglycemia, hyperlipidemia, hyperuricemia, hepatomegaly, and nephromegaly mimicking the abnormal metabolic phenotype of human GSD-Ia. We then treated newborn G6PC-R83C mice with lipid nanoparticles encompassing the guide RNA and mRNA encoding ABE (LNP-ABE) and showed that the treated mice grew normally to age 8 weeks without hypoglycemia seizures. The LNP-ABE-treated G6PC-R83C mice expressed significant levels of hepatic G6Pase- activity with an editing efficiency up to 60% and displayed normalized blood metabolite profiles and could tolerate 24 hours of fasting. Taken together, our data demonstrate the potential of base-editing to correct the G6PC-p.R83C mutation in its native genetic locus could lead to potentially permanent, durable, long-term correction of the GSD-Ia disorder. GSD-Ia patients manifest nephromegaly caused by marked glycogen accumulation and nephropathy. The current dietary therapies have significantly alleviated metabolic abnormalities and delayed chronic renal disease and renal insufficiency in GSD-Ia patients. However, the underlying pathological processes remain uncorrected, glomerular hyperfiltration, hypercalciuria, hypocitraturia, and urinary albumin excretion still occur in metabolically compensated GSD-Ia patients. We have shown that one mechanism that underlies GSD-Ia nephropathy is fibrosis mediated by activation of the renin-angiotensin system (RAS). The Wnt/-catenin signaling that promotes fibrosis controls the expression of RAS genes. We hypothesized that elevated renal glycogen could elicit acute kidney injury (AKI) that activates Wnt/-catenin signaling and promotes fibrosis. Here we show that G6pc-/- mice displayed impaired renal glucose homeostasis and AKI. Renal levels of -catenin increased markedly in G6pc-/- mice during postnatal development, along with elevated renal levels of renin, angiotensinogen, and snail1. Renal fibrosis was evident by increased renal levels of -smooth muscle actin (-SMA) and extracellular matrix (ECM) proteins. ICG-001, a -catenin inhibitor, reduced renal levels of renin, snail1, -SMA, and ECM proteins, indicating that targeting the Wnt/-catenin s

当前I/II期临床试验中使用的rAAV-co-G6PC载体是附加型表达的，目前正在确定在人类中表达的长期持久性。因此，我们试图探索使用 CRISPR/Cas9 技术来纠正其天然基因座中的致病性 GSD-Ia 变异。在白种人 GSD-Ia 患者中发现的最常见的致病突变是 G6PC-p.R83C，占患病等位基因的 32%。利用基于 CRISPR/Cas9 的基因编辑技术，我们生成了 GSD-Ia 小鼠疾病模型，即 G6PC-p.R83C 突变纯合子 G6pc-R83C 小鼠，并表明 G6pc-R83C 小鼠表现出类似于人类 GSD-Ia。然后，我们使用基于 CRISPR/Cas9 的基因编辑系统治疗新生 G6pc-R83C 小鼠，结果显示接受治疗的小鼠正常生长至 16 周，没有出现低血糖发作。接受治疗的 G6pc-R83C 小鼠表达正常肝脏 G6Pase 活性的 3%，维持葡萄糖稳态，显示正常化的血液代谢，并且可以维持 24 小时禁食。总而言之，我们开发了第二代疗法，在体内纠正其天然基因座中的致病性 G6PC-p.R83C 变异可能会导致 GSD-Ia 疾病的潜在永久、持久、长期纠正。 临床上，GSD-1b患者表现出血糖稳态受损的代谢表型和肝细胞腺瘤/癌（HCA/HCC）的长期风险。 GSD-Ib 中 HCA/HCC 的病因尚不清楚。研究表明，自噬是一种进化保守的降解过程，在营养匮乏和环境压力下，通过溶酶体降解细胞内蛋白质和细胞器产生能量和结构单元，自噬的缺乏会导致肝癌的发生。自噬可通过 Sirtuin 1 (SIRT1)、AMP 激活蛋白激酶 (AMPK) 和叉头盒 O (FoxO) 转录因子家族成员进行正向调节。在肝脏中，AMPK 通过肝激酶 B-1 (LKB1)（一种丝氨酸/苏氨酸激酶）磷酸化残基 T172 处的 AMPK 亚基而被激活。为了了解 GSD-Ib 肝癌发生的途径，我们假设受损的肝自噬是一个重要因素。在这项研究中，我们发现 G6PT 缺乏会导致肝自噬受损，这从自噬网络许多组件的表达减弱、自噬体形成减少和自噬通量减少中可以明显看出。 G6PT 缺陷的肝脏表现出 SIRT1 和 AMPK 信号传导受损，以及 SIRT1、FoxO3a、LKB1 和活性 p-AMPK 表达减少。重要的是，我们发现在 G6PT 缺陷的肝脏中过度表达 SIRT1 或 LKB1 可以恢复自噬以及 SIRT1/FoxO3a 和 LKB1/AMPK 信号传导。 G6PT 缺陷肝脏中的肝脂肪变性降低了 SIRT1 的表达。 LKB1 过表达降低了肝甘油三酯水平，提供了 LKB1/AMPK 信号上调与 SIRT1 表达增加之间的潜在联系。总之，SIRT1/FoxO3a 和 LKB1/AMPK 信号传导的下调是肝自噬受损的基础，这可能有助于 GSD-Ib 中 HCA/HCC 的发展。了解这一机制可能会指导未来的治疗。 我们已经生成了4个有效的G6PC基因转移rAAV载体，用于GSD-Ia基因治疗； rAAV-G6PC 表达野生型 (WT) G6PC，rAAV-coG6PC 表达密码子优化 (co) G6PC，rAAV-G6PC-S298C 表达功效增强的 G6PC-S298C 变体，以及 rAAV-coG6PC-S298C。我们的 rAAV-G6PC/rAAV-coG6PC 载体（美国专利#9,644,216）技术已授权给 Ultragenyx Pharmaceutical Inc，该公司已于 2018 年启动 I/II 期临床试验（NCT03517085），并于 2022 年启动 III 期临床试验（NCT05139316）使用 rAAV-GPE-coG6PC 载体。为了检验这些 rAAV 载体的长期功效，我们使用这些 rAAV 载体在 G6pc-/- 小鼠中进行了长期（66-76 周）基因转移研究。所有接受治疗的 G6pc-/- 小鼠均存活至 66-76 周，且结果具有累加性。 rAAV-G6PC-S298C-、rAAV-coG6PC- 和 rAAV-coG6PC-S298C- 处理的 G6pc-/- 小鼠的肝脏 G6Pase 活性分别比 rAAV-G6PC 的 1.7、1.7 和 4.4 倍高-WT处理的小鼠。 rAAV-coG6PC-S298C载体的功效比目前用于III期临床试验的rAAV-coG6PC载体（NCT05139316）高2.6倍。总而言之，rAAV-G6PC-S298C 和 rAAV-coG6PC-S298C 载体提供了有吸引力的临床替代方案。 我们探索基于腺嘌呤碱基编辑器 (ABE) 的技术，该技术能够实现基因组 DNA 中 AT 到 GC 的可编程转换，用于 GSD-Ia 疗法。 ABE 系统适用于分裂和非分裂细胞，据报道在基因组中几乎不产生插入缺失或脱靶编辑，可以纠正其天然基因位点中的致病性变异，从而导致永久的、治疗有效的长期表达。这是与马萨诸塞州剑桥 Beam Therapeutics 在 CRADA 下进行的一项合作研究。 G6PC-p.R83C 是在高加索 GSD-Ia 患者中发现的最常见的致病性突变，其在 G6PC 基因中包含单个 G>A 转变。我们首先通过将人 G6PC-p.R83C 的完整编码序列以及人 G6PC 3-UTR 插入小鼠 G6pc 基因的外显子 1 的 ATG 起始处，生成纯合人源化 R83C/R83C 小鼠品系，即 G6PC-R83C 小鼠密码子。该插入将人类转录物置于天然小鼠 G6pc 启动子/增强子的控制之下。小鼠 G6pc 基因被小鼠 G6pc 外显子 1 中产生的过早终止密码子破坏。我们发现 G6PC-R83C 小鼠表现出葡萄糖稳态受损，其特征是生长迟缓、低血糖、高脂血症、高尿酸血症、肝肿大和肾肿大，类似于代谢异常。人类 GSD-Ia 的表型。然后，我们用脂质纳米颗粒治疗新生 G6PC-R83C 小鼠，该纳米颗粒包含指导 RNA 和编码 ABE 的 mRNA (LNP-ABE)，结果表明，经过治疗的小鼠正常生长至 8 周龄，没有出现低血糖发作。 LNP-ABE 处理的 G6PC-R83C 小鼠表达显着水平的肝脏 G6Pase 活性，编辑效率高达 60%，并显示标准化的血液代谢特征，并且可以耐受 24 小时禁食。总而言之，我们的数据证明了碱基编辑纠正其天然基因位点中的 G6PC-p.R83C 突变的潜力，可能会导致 GSD-Ia 疾病的潜在永久、持久、长期纠正。 GSD-Ia患者表现出由明显的糖原积累和肾病引起的肾肿大。目前的饮食疗法已显着减轻了GSD-Ia患者的代谢异常并延缓了慢性肾病和肾功能不全的发生。然而，潜在的病理过程仍未得到纠正，代谢代偿的 GSD-Ia 患者仍然出现肾小球高滤过、高钙尿、低柠檬酸尿和尿白蛋白排泄。 我们已经证明，GSD-Ia 肾病的一种机制是肾素-血管紧张素系统 (RAS) 激活介导的纤维化。促进纤维化的 Wnt/-连环蛋白信号传导控制 RAS 基因的表达。我们假设肾糖原升高可能引发急性肾损伤 (AKI)，从而激活 Wnt/-连环蛋白信号传导并促进纤维化。在这里，我们发现 G6pc-/- 小鼠表现出肾葡萄糖稳态受损和 AKI。 G6pc-/- 小鼠在出生后发育过程中，肾脏的β-连环蛋白水平显着增加，同时肾素、血管紧张素原和 snail1 的肾脏水平也升高。肾纤维化通过肾内β-平滑肌肌动蛋白（-SMA）和细胞外基质（ECM）蛋白水平的增加而明显可见。 ICG-001 是一种 β-连环蛋白抑制剂，可降低肾素、snail1、-SMA 和 ECM 蛋白的肾水平，表明靶向 Wnt/-连环蛋白