Molecular Genetics Of Heritable Human Disorders

人类遗传性疾病的分子遗传学

基本信息

批准号：
10901680
负责人：
JANICE CHOU
金额：
$ 194.22万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/10901680
关键词：
3&apos Splice Site 5&apos -AMP-activated protein kinase Actins Active Sites Acute Renal Failure with Renal Papillary Necrosis Adenine Age Albumins Animal Model Attenuated Autophagocytosis Autophagosome Blood Blood Glucose CRISPR therapeutics CRISPR/Cas technology Carcinoma Caucasians Childhood Chronic Kidney Failure Clinical Clinical Data Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Code Collaborations Collagen Collagen Type IV Compensation Complex Complication Cooperative Research and Development Agreement Couples Cytoplasm Data Development Diet therapy Disease Dose Down-Regulation Endoplasmic Reticulum Enhancers Etiology Excretory function Exhibits Exons Extracellular Matrix Proteins Face Family member Fasting Fatty Liver Fibrosis Functional disorder Future Genes Genetic Diseases Genetic Transcription Genome Genomic DNA Gluconeogenesis Glucose Glucose-6-Phosphate Glycogen Glycogen Storage Disease Type I Goals Growth Guide RNA Hepatic Hepatocarcinogenesis Hepatomegaly Heritability Homeostasis Hour Human Hydrolysis Hyperlipidemia Hyperuricemia Hypoglycemia Impairment Initiator Codon Inorganic Phosphate Transporter Integral Membrane Protein Interphase Cell Intestines Kidney Kidney Diseases Laboratories Legal patent Lesion Licensing Link Liver Liver Cell Adenoma Mediating Mediator Messenger RNA Metabolic Molecular Molecular Genetics Mouse Strains Mus Mutation Neonatal Newborn Infant Nonhomologous DNA End Joining Nonsense Codon Nuclear Translocation Oligonucleotides Organelles Outcome Pathogenicity Pathologic Processes Pathway interactions Patients Pharmacologic Substance Phase Phase I/II Clinical Trial Phase III Clinical Trials Phenotype Phosphorylation Phosphotransferases Process Protein-Serine-Threonine Kinases Proteins Proximal Kidney Tubules RNA Splicing Reagent Recombinant adeno-associated virus (rAAV)Renin Renin-Angiotensin System Reporting Risk SIRT1 gene Seizures Serine Signal Transduction Smooth Muscle Snails System Technology Therapeutic Transcript Triglycerides Untranslated Regions Up-Regulation Variant Work adeno-associated viral vector base editing base editor blood glucose regulation ds-DNA efficacy evaluation environmental stressor gene augmentation therapy gene correction gene therapy genetic technology genomic locus glucose production glucose-6-phosphatase hypercalciuria inhibitor inorganic phosphate insertion/deletion mutation kidney fibrosis lipid nanoparticle metabolic phenotype novel therapeutic intervention novel therapeutics nutrient deprivation overexpression preclinical study promoter renal damage repair strategy repaired therapeutically effective transcription factor transgene expression urinary vector virtual

项目摘要

Clinically, GSD-Ib patients manifest a metabolic phenotype of impaired blood glucose homeostasis and a 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. GSD-Ia is a pediatric genetic disorder. The rAAV-G6PC vector used in Phase III clinical trial for GSD-Ia (NCT05139316) is episomally expressed. Currently, there is insufficient clinical data to understand if multi-decade episomal transgene expression can be maintained in the human liver at a therapeutic level. We therefore explored alternative genetic technologies for GSD-Ia therapy, such as CRISPR/Cas9-based gene editing. We previously generated a G6pc-R83C mouse strain carrying the prevalent pathogenic G6PC-p.R83C variant and showed that the G6pc-R83C mice exhibit the pathophysiology of impaired glucose homeostasis mimicking human GSD-Ia. In an initial exploration of CRISP/Cas-9-based editing using AAV to deliver the CRISPR reagents, we showed that a homology directed repair strategy could correct the abnormal metabolic phenotype of neonatal G6pc-R83C mice. Using the G6pc-R83C mice, we now explored a CRISPR/Cas9-based double-strand DNA oligonucleotide (dsODN) insertional strategy that uses the non-homologous end joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon-2. The strategy is based on the insertion of a short dsODN into G6pc exon-2 to disrupt the native exon, and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced the edited gene would generate a wild-type mRNA encoding the native G6Pase- protein. The editing reagents formulated in lipid nanoparticles (LNP) were delivered to the liver. Mice were treated either with one dose of LNP-dsODN at age 4 weeks or with 2 doses of LNP-dsODN at age 2 and 4 weeks. The G6pc-R83C mice receiving successful editing expressed 4% of normal hepatic G6Pase- activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD-Ia phenotype. Renal disease is a serious long-term complication for GSD-Ia. The early kidney manifestations of GSD-Ia are impaired renal gluconeogenesis, and nephromegaly caused by increased glycogen accumulation. The only therapies currently available to treat GSD-Ia are dietary therapies which have significantly alleviated metabolic abnormalities but only delay the onset of chronic kidney disease. The underlying pathological processes remain uncorrected, and glomerular hyperfiltration, hypercalciuria, hypocitraturia and urinary albumin excretion still occur in metabolically compensated GSD-Ia patients. We have previously shown that one mechanism underlies GSD-Ia nephropathy is fibrosis mediated by activation of the renin-angiotensin system (RAS). The Wnt/-catenin signaling regulates the expression of a variety of downstream mediators implicated in renal fibrosis, including multiple genes in the RAS. Sustained activation of Wnt/-catenin signaling is associated with the development and progression of renal fibrotic lesions that can lead to chronic kidney disease. In this study, we examined the molecular mechanism underlying GSD-Ia nephropathy. Damage to the kidney proximal tubules is known to trigger acute kidney injury (AKI) that can, in turn, activate Wnt/-catenin signaling. We show that GSD-Ia mice display AKI that leads to activation of the Wnt/-catenin/RAS axis. Renal fibrosis was demonstrated by increased renal levels of Snail1, -smooth muscle actin (-SMA), and extracellular matrix proteins, including collagen-I1 and collagen-IV. Treating GSD-Ia mice with a CBP/-catenin inhibitor, ICG-001, significantly decreased nuclear translocated active -catenin and reduced renal levels of renin, Snail1, -SMA, and collagen-IV. The results suggest that inhibition of Wnt/-catenin signaling may be a promising therapeutic strategy for GSD-Ia nephropathy. We explore the Adenine base editor (ABE)-based technologies that enable a programmable conversion of AT to GC in genomic DNA for GSD-Ia therapy. 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 (LNP) 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

临床上，GSD-1b患者表现出血糖稳态受损的代谢表型以及肝细胞腺瘤/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。 GSD-Ia 是一种儿科遗传性疾病。 GSD-Ia III 期临床试验中使用的 rAAV-G6PC 载体 (NCT05139316) 是附加型表达的。目前，没有足够的临床数据来了解是否可以在人肝脏中将数十年的附加转基因表达维持在治疗水平。因此，我们探索了 GSD-Ia 疗法的替代基因技术，例如基于 CRISPR/Cas9 的基因编辑。我们之前生成了携带普遍致病性 G6PC-p.R83C 变体的 G6pc-R83C 小鼠品系，并表明 G6pc-R83C 小鼠表现出模仿人类 GSD-Ia 的葡萄糖稳态受损的病理生理学。在使用 AAV 传递 CRISPR 试剂的基于 CRISP/Cas-9 的编辑的初步探索中，我们表明同源定向修复策略可以纠正新生 G6pc-R83C 小鼠的异常代谢表型。使用 G6pc-R83C 小鼠，我们现在探索了一种基于 CRISPR/Cas9 的双链 DNA 寡核苷酸 (dsODN) 插入策略，该策略使用非同源末端连接修复机制来纠正 G6pc 外显子 2 中的致病性 p.R83C 变异。该策略基于将短 dsODN 插入 G6pc 外显子 2 以破坏天然外显子，并引入额外的剪接受体位点和校正序列。当转录和剪接时，编辑的基因将产生编码天然 G6Pase 蛋白的野生型 mRNA。以脂质纳米粒子（LNP）配制的编辑试剂被递送至肝脏。小鼠在 4 周龄时接受一剂 LNP-dsODN 治疗，或在 2 周龄和 4 周龄时接受 2 剂 LNP-dsODN 治疗。接受成功编辑的 G6pc-R83C 小鼠表达正常肝脏 G6Pase 活性的 4%，维持葡萄糖稳态，没有低血糖癫痫发作，并表现出正常化的血液代谢谱。结果与支持之前正在进行临床试验的基因增强疗法的临床前研究一致。这种编辑策略可能为比基因增强更早进行临床干预的治疗方法提供基础，并具有潜在永久纠正 GSD-Ia 表型的额外好处。肾脏疾病是 GSD-Ia 的严重长期并发症。 GSD-Ia的早期肾脏表现是肾糖异生受损，以及糖原积累增加引起的肾肿大。目前唯一可用于治疗 GSD-Ia 的疗法是饮食疗法，它可以显着减轻代谢异常，但只能延缓慢性肾病的发作。潜在的病理过程仍未得到纠正，在代谢代偿的 GSD-Ia 患者中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。我们之前已经表明，GSD-Ia 肾病的一种机制是由肾素-血管紧张素系统 (RAS) 激活介导的纤维化。 Wnt/-连环蛋白信号传导调节与肾纤维化有关的多种下游介质的表达，包括 RAS 中的多个基因。 Wnt/-连环蛋白信号传导的持续激活与肾纤维化病变的发生和进展相关，肾纤维化病变可导致慢性肾病。在这项研究中，我们研究了 GSD-Ia 肾病的分子机制。已知肾脏近曲小管损伤会引发急性肾损伤 (AKI)，进而激活 Wnt/-连环蛋白信号传导。我们发现 GSD-Ia 小鼠表现出 AKI，导致 Wnt/-catenin/RAS 轴激活。肾纤维化表现为肾 Snail1、β-平滑肌肌动蛋白 (-SMA) 和细胞外基质蛋白（包括胶原蛋白-I1 和胶原蛋白-IV）水平升高。用 CBP/-连环蛋白抑制剂 ICG-001 治疗 GSD-Ia 小鼠，显着降低核转位的活性-连环蛋白，并降低肾素、Snail1、-SMA 和胶原蛋白-IV 的肾水平。结果表明，抑制 Wnt/-catenin 信号传导可能是 GSD-Ia 肾病的一种有前途的治疗策略。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 治疗。 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（LNP）治疗新生 G6PC-R83C 小鼠，该纳米颗粒包含引导 RNA 和编码 ABE 的 mRNA (LNP-ABE)，结果显示，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 G6PC-p.R83C 突变的潜力

项目成果

期刊论文数量（42）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structure-function study of the glucose-6-phosphate transporter, an eukaryotic antiporter deficient in glycogen storage disease type Ib.

DOI：
10.1016/j.ymgme.2008.10.005
发表时间：
2009-01
期刊：
MOLECULAR GENETICS AND METABOLISM
影响因子：
3.8
作者：
Pan, Chi-Jiunn;Chen, Shih-Yin;Lee, Soojung;Chou, Janice Y.
通讯作者：
Chou, Janice Y.

The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia.

DOI：
10.1016/j.ymgme.2013.06.014
发表时间：
2013-11
期刊：
MOLECULAR GENETICS AND METABOLISM
影响因子：
3.8
作者：
Lee, Young Mok;Pan, Chi-Jiunn;Koeberl, Dwight D.;Mansfield, Brian C.;Chou, Janice Y.
通讯作者：
Chou, Janice Y.

Downregulation of SIRT1 signaling underlies hepatic autophagy impairment in glycogen storage disease type Ia.

DOI：
10.1371/journal.pgen.1006819
发表时间：
2017-05
期刊：
PLoS genetics
影响因子：
4.5
作者：
Cho JH;Kim GY;Pan CJ;Anduaga J;Choi EJ;Mansfield BC;Chou JY
通讯作者：
Chou JY

Normoglycemia alone is insufficient to prevent long-term complications of hepatocellular adenoma in glycogen storage disease type Ib mice.

DOI：
10.1016/j.jhep.2008.11.026
发表时间：
2009-11
期刊：
JOURNAL OF HEPATOLOGY
影响因子：
25.7
作者：
Yiu, Wai Han;Pan, Chi-Jiunn;Mead, Paul A.;Starost, Matthew F.;Mansfield, Brian C.;Chou, Janice Y.
通讯作者：
Chou, Janice Y.

Mutations in the glucose-6-phosphatase-alpha (G6PC) gene that cause type Ia glycogen storage disease.