Yasuda Makiko, Domaradzki Maciej E, Armentano Donna, Cheng Seng H, Bishop David F, Desnick Robert J
Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA.
J Gene Med. 2007 Sep;9(9):806-11. doi: 10.1002/jgm.1074.
Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by the half-normal activity of hydroxymethylbilane synthase (HMB-synthase). Affected individuals can experience episodic, life-threatening, acute neurological attacks that are precipitated by various drugs, dieting, and hormonal changes. Intravenous hematin is used to treat the attacks, but a more effective, preventive therapy is needed, especially for patients with frequent attacks. Since the disease is a hepatic encephalopathy, efforts were focused towards evaluating four different combinations of liver-specific enhancers and promoters for maximal hepatic HMB-synthase expression.
Four different mammalian expression vectors, each carrying a unique combination of liver-specific enhancers and promoters driving murine HMB-synthase cDNA expression, were transiently transfected into HepG2 cells. The vectors included: HMBS-1; human alpha1-microglobulin enhancer/alpha1-antityrpsin promoter (alpha1Me/alpha1ATp), HMBS-2; alpha1Me/human serum albumin promoter (alpha1Me/SAp), HMBS-3; human prothrombin enhancer/SAp (PTe/SAp), and HMBS-4; (PTe/alpha1ATp). Each HMB-synthase construct and a luciferase reporter construct were hydrodynamically coinjected into mice with HMB-synthase deficiency and evaluated for hepatic expression 24 h post-injection, the time-point of peak hepatic HMB-synthase expression.
Following transient transfection into HepG2 cells, HMBS-1 (alpha1Me/alpha1ATp) had the highest HMB-synthase expression level, with an approximately 8-fold increase over endogenous cellular activities. Construct HMBS-1 also had the highest hepatic HMB-synthase activity following hydrodynamic delivery into HMB-synthase deficient mice, with a approximately 6-fold increase over saline-treated mice.
These studies support the use of a gene therapy vector containing the alpha1Me/alpha1ATp combination for preclinical studies of the efficacy and safety of liver-targeted gene therapy for AIP.
急性间歇性卟啉病（AIP）是一种常染色体显性疾病，由羟甲基胆色素原合酶（HMB - 合酶）活性减半所致。患者可能会经历由各种药物、节食和激素变化引发的发作性、危及生命的急性神经发作。静脉注射血红素用于治疗发作，但需要一种更有效、预防性的疗法，尤其是对于频繁发作的患者。由于该疾病是一种肝性脑病，研究重点在于评估四种不同的肝脏特异性增强子和启动子组合，以实现肝脏中HMB - 合酶的最大表达。
将四种不同的哺乳动物表达载体瞬时转染至HepG2细胞，每个载体携带驱动小鼠HMB - 合酶cDNA表达的独特肝脏特异性增强子和启动子组合。这些载体包括：HMBS - 1；人α1 - 微球蛋白增强子/α1 - 抗胰蛋白酶启动子（α1Me/α1ATp），HMBS - 2；α1Me/人血清白蛋白启动子（α1Me/SAp），HMBS - 3；人凝血酶原增强子/SAp（PTe/SAp），以及HMBS - 4；（PTe/α1ATp）。将每种HMB - 合酶构建体和荧光素酶报告基因构建体通过流体动力学方法共注射到HMB - 合酶缺陷小鼠体内，并在注射后24小时（肝脏HMB - 合酶表达峰值的时间点）评估肝脏表达情况。
瞬时转染至HepG2细胞后，HMBS - 1（α1Me/α1ATp）具有最高的HMB - 合酶表达水平，比内源性细胞活性增加约8倍。在通过流体动力学方法导入HMB - 合酶缺陷小鼠后，构建体HMBS - 1也具有最高的肝脏HMB - 合酶活性，比盐水处理的小鼠增加约6倍。
这些研究支持使用含有α1Me/α1ATp组合的基因治疗载体，用于AIP肝脏靶向基因治疗疗效和安全性的临床前研究。
