Gene delivery to striated muscle by systemic AAV vectors

描述(由申请人提供)： 糖原沉积病II(GsD-II；Pompe病；MiM 232300)是由于酸性α-葡萄糖苷酶(GAA)缺乏引起的一种致死性肌营养不良症。GSD-II尚无根治疗法。GSD-II基因治疗的障碍包括非常高的GAA要求和中和抗GAA抗体的发生。腺相关病毒(AAV)载体的基因治疗是随着AAV血清型6-9的出现而发展起来的，在这一应用中被称为肌营养型AAV载体。一般的假设是，肌肉营养型AAV载体将在GSD-II小鼠的横纹肌中表达有效的GAA，包括心脏、横纹肌和骨骼肌。假型为AAV7、AAV8和AAV9的肌肉营养型AAV2载体将被静脉注射，以分析这些载体对横纹肌的全身输送。特定假设1：含有肌肉特异性启动子的假型AAV载体将在GSD-II小鼠的横纹肌中表达高水平的GAA。针对GAA基因敲除(GAA-KO)小鼠的每种血清型，将对心脏、横隔膜和骨骼肌中GAA缺乏的纠正和糖原储存进行分析。肌肉营养型AAV载体将被应用于婴儿GAA-KO小鼠，展示其对庞贝病的疗效。特定假设2：AAV载体将逃避细胞和体液免疫反应，以实现对GSD-II小鼠的长期疗效。对仅限于横纹肌的HGAA表达的细胞和体液免疫反应进行分析。ELISPOT试验将被用来分析抗原特异性T细胞对HGAA和AAV衣壳的反应，而其他淋巴细胞标志物的流式细胞仪检测将评估对AAV载体的免疫反应。特异性假说3：肝脏特异性表达将诱导GSD-II小鼠对引入的GAA的耐受性，并增强肌肉营养型AAV载体的疗效。在具有免疫功能的GAA-KO小鼠中，肝脏特异性表达GAA将诱导对GAA的耐受。在耐受的GAA-KO小鼠中，T调节细胞将被定量；此外，T调节细胞将被耗尽，以证明它们在建立对GAA的耐受性中的作用。一种新的策略将通过静脉注射和隔离肢体递送的方式传递编码嵌合GAA的AAV载体，以将骨骼肌转化为分泌GAA的仓库。GSD-II中有效的肌肉靶向基因治疗将对其他肌肉营养不良和肌病的基因治疗具有指导意义。

DESCRIPTION (provided by applicant): Glycogen storage disease II (GSD-II; Pompe disease; MIM 232300) is a lethal muscular dystrophy caused by the deficiency of acid alpha-glucosidase (GAA; acid maltase). No curative therapy is available for GSD-II. Obstacles to gene therapy in GSD-II include very high GAA requirements and the occurence of neutralizing anti-GAA antibodies. Gene therapy with adeno-associated virus (AAV) vectors was advanced by the availability of AAV serotypes 6-9, termed myotrophic AAV vectors in this application. The general hypothesis states that myotrophic AAV vectors will express efficacious GAA in the striated muscles of GSD-II mice, including the heart, diaphragm, and skeletal muscles. Myotrophic AAV2 vectors pseudotyped as AAV7, AAV8, and AAV9 will be delivered intravenously to analyze the systemic delivery of these vectors to striated muscle. Specific Hypothesis 1: Pseudotyped AAV vectors containing a muscle-specific promoter will express high levels of GAA in the striated muscle of GSD-II mice. Correction of GAA deficiency and glycogen storage in the heart, diaphragm, and skeletal muscle will be analyzed for each serotype in GAA- knockout (GAA-KO) mice. Myotrophic AAV vectors will be administered to infantile GAA-KO mice, demonstrating efficacy in Pompe disease. Specific Hypothesis 2: An AAV vector will evade cellular and humoral immune responses to achieve long-term efficacy in GSD-II mice. Cellular and humoral immune responses to hGAA expression restricted to striated muscles will be analyzed. ELISpot assays will be performed to analyze antigen-specific T cell responses to hGAA and AAV capsids, and flow cytometric detection of additional lymphocyte markers will evaluate the immune response to AAV vectors. Specific Hypothesis 3: Liver-specific expression will Induce tolerance to introduced GAA and enhance the efficacy of myotrophic AAV vectors in GSD-II mice. Tolerance to GAA will be induced by liver-specific expression of GAA in immunocompetent GAA-KO mice. T regulatory cells will be quantitated in tolerant GAA-KO mice; furthermore, T regulatory cells will be depleted to demonstrate their role in establishing tolerance to GAA. A novel strategy will deliver an AAV vector encoding chimeric GAA by intravenous injection and isolated limb delivery to convert skeletal muscle to a depot for secreted GAA. Efficacious muscle-targeted gene therapy in GSD-II will have implications for gene therapy in other muscular dystrophies and myopathies.