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

IX型肝糖原储存疾病的总体估计患病率为每10万人中有1人，约占所有GSD病例的25%。肝脏GSD IX是由肝酶磷酸酶激酶(PhK)缺乏引起的，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，由四个亚基组成-α，β，γ和δ-每个亚基都有不同基因编码的组织特异性亚型。PHKA2、PHKB和PHKG2基因分别编码肝脏特异性亚基α-2、β和γ-2。直到最近基因面板和外显子组测序的出现，肝脏GSD IX的诊断还不能区分这些亚型。越来越多的证据表明，第二种最常见的亚型PHKG2 GSDIX(GSDIXγ2)的患者会患上严重的肝病。在已发表的病例报告中，95.8%患有GSD IXγ2的患者报告有肝纤维化和/或肝硬变的特征，这使患有GSD IXγ2的患者面临更高的肝功能衰竭、肝细胞癌和死亡风险。尽管GSD IXγ2严重威胁生命，但对了解疾病进展或治疗选择的研究一直很少。本项目的第一个目标是鉴定第一个PHKG2 GSD IX(GSD IXγ2)小鼠模型的表型。来自3个月大的雄性和雌性Phkg2-/-小鼠的初步证据令人鼓舞。我发现，与野生型对照相比，基因敲除小鼠的肝糖原含量、血清ALP、AST、ALT、尿Hex4以及肝细胞肿大和纤维化的特征都显著升高。我将继续研究6个月、9个月和12个月大的Phkg2-/-小鼠的表型。通过在我们的模型中确定肝病的发病和进展时间，我将更好地理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的AAV基因治疗方法在Phkg2-/-小鼠模型中减缓肝病进展的有效性。目前的肝脏导向基因治疗利用重组腺相关病毒血清8型(AAV8)，基于小鼠的临床前疗效。然而，最近的研究表明，AAV8对小鼠肝细胞比对人肝细胞有更大的趋向性。我们的团队已经确定了一种对人和小鼠肝细胞都具有高转导率的新型衣壳蛋白(AAVhum.8)，使其成为临床前评估的优秀载体，具有很高的临床翻译潜力。该项目的结果将表征首个GSD IXγ2小鼠模型，将为GSD IXγ2患者的非手术、长期治疗方案提供临床前证据，并将为其他儿童遗传性肝病的治疗提供参考。

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.