Liver-directed AAV gene therapy for PHKG2-Glycogen Storage Disease IX (GSD IX y2)

肝糖原累积病IX型的总体估计患病率为1/100，000，约占所有GSD病例的25%。肝GSD IX是由肝酶磷酸化酶激酶（PhK）缺乏引起的，并表现为肝肿大、肝酶升高和低血糖。PhK是一种复杂的异源四聚体酶，由四个亚基- α、β、γ和δ -组成，每个亚基具有由不同基因编码的组织特异性同种型。基因PHKA 2、PHKB和PHKG 2分别编码肝脏特异性同种型PhK亚基α2、β和γ2。直到最近可用的基因面板和外显子组测序，肝GSD IX的诊断不允许区分这些亚型。越来越多的证据表明，患有第二种最常见亚型PHKG 2 GSD IX（GSD IX γ2）的患者会发生严重的肝脏疾病。在已发表的病例报告中，95.8%的GSD IX γ2患者报告了肝纤维化和/或肝硬化的特征，使GSD IX γ2患者的肝衰竭、肝细胞癌和死亡风险更高。尽管GSD IX γ2的严重程度危及生命，但了解疾病进展或治疗选择的研究很少。该项目的第一个目标是表征PHKG 2 GSD IX（GSD IX γ2）的第一个小鼠模型的表型。来自雄性和雌性3个月大Phkg 2-/-小鼠的初步证据令人鼓舞。我已经发现，与野生型对照相比，基因敲除小鼠具有显著升高的肝糖原含量、血清ALP、AST、ALT、尿Hex 4以及肝细胞增大和纤维化的特征。我将继续在6、9和12月龄时表征Phkg 2-/-小鼠表型。通过确定我们模型中肝病的发作和进展时间，我将更好地了解提供治疗的理想时间。该项目的第二个目标是评估一种新型AAV基因治疗方法在Phkg 2-/-小鼠模型中减少肝脏疾病进展的疗效。目前的肝脏定向基因疗法利用重组腺相关病毒血清型8（AAV 8），基于在小鼠中的临床前功效。然而，最近的研究已经证明，AAV 8对鼠肝细胞比对人肝细胞具有更大的向性。我们的研究小组已经确定了一种对人和鼠肝细胞都具有高转导率的新型衣壳（AAVhum.8）-使其成为具有高临床转化潜力的临床前评价的优秀载体。该项目的结果将表征第一个GSD IX γ2小鼠模型，将为GSD IX γ2患者的非手术，长期治疗选择提供临床前证据，并将为其他儿科遗传性肝病的治疗提供信息。

Liver Glycogen Storage Disease type IX has an overall estimated prevalence of 1 in 100,000 individuals, accounting for approximately 25% of all GSD cases. Liver GSD IX is caused by deficiency of the liver enzyme phosphorylase kinase (PhK) and presents with hepatomegaly, elevated liver enzymes, and hypoglycemia. PhK is a complex, hetero-tetrameric enzyme comprised of four subunits - α, β, γ, and δ - each with tissue specific isoforms encoded by different genes. The genes PHKA2, PHKB, and PHKG2 encode the liver specific isoform PhK subunits α2, β, and γ2 respectively. Until the recent availability of gene panels and exome sequencing, the diagnosis of Liver GSD IX did not allow for differentiation of these subtypes. There is growing evidence that patients with the second most common subtype, PHKG2 GSD IX (GSD IX γ2) develop severe liver disease. Of published case reports, 95.8% of patients with GSD IX γ2 reported features of liver fibrosis and/or cirrhosis, placing individuals with GSD IX γ2 at higher risk for liver failure, hepatocellular carcinoma and death. Despite the life-threatening severity of GSD IX γ2, there has been minimal research to understand disease progression or options for treatment. The first goal of this project is to characterize the phenotype of the first mouse model of PHKG2 GSD IX (GSD IX γ2). Preliminary evidence from male and female 3-month-old Phkg2-/- mice is encouraging. I have discovered that knockout mice have significantly elevated liver glycogen content, serum ALP, AST, ALT, urine Hex4, and features of hepatocyte enlargement and fibrosis compared to wild type controls. I will continue to characterize the Phkg2-/- mouse phenotype at 6, 9- and 12-months of age. By identifying the time of onset and progression of liver disease in our model, I will better understand the ideal time to deliver therapy. The second goal of this project is to evaluate the efficacy of a novel AAV gene therapy approach for reducing liver disease progression in the Phkg2-/- mouse model. Current liver-directed gene therapies utilize a recombinant adeno-associated virus serotype 8 (AAV8), based on preclinical efficacy in mice. However, recent studies have demonstrated that AAV8 has greater tropism for murine versus human hepatocytes. Our group has identified a novel capsid with high transduction rates for both human and murine hepatocytes (AAVhum.8) – making it an excellent vector for preclinical evaluation with high potential for clinical translation. The results of this project will characterize the first GSD IX γ2 mouse model, will provide preclinical evidence for a non-surgical, long-term, therapeutic option for patients with GSD IX γ2, and will inform the treatment of other pediatric genetic liver diseases.