Gene delivery to striated muscle by systemic AAV vectors

描述（由申请人提供）： 糖原累积病II（GSD-II;庞贝氏症; MIM 232300）是由酸性α-葡糖苷酶（GAA;酸性麦芽糖酶）缺乏引起的致死性肌营养不良症。GSD-II没有治愈性治疗。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）载体的基因治疗由于AAV血清型6-9（在本申请中称为肌营养型AAV载体）的可用性而得到了发展。一般假设表明肌营养型AAV载体将在GSD-II小鼠的横纹肌（包括心脏、横膈膜和骨骼肌）中表达有效的GAA。将假型化为AAV 7、AAV 8和AAV 9的肌营养型AAV 2载体静脉内递送以分析这些载体向横纹肌的全身递送。具体假设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。将分析GAA敲除（GAA-KO）小鼠中每种血清型的心脏、膈肌和骨骼肌中GAA缺乏和糖原储存的校正。将肌营养型AAV载体施用给婴儿GAA-KO小鼠，证明在庞贝氏症中的功效。特定假设2：AAV载体将逃避细胞和体液免疫应答，以在GSD-II小鼠中实现长期功效。将分析对限于横纹肌的hGAA表达的细胞和体液免疫应答。将进行ELISpot测定以分析对hGAA和AAV衣壳的抗原特异性T细胞应答，并且另外的淋巴细胞标志物的流式细胞术检测将评价对AAV载体的免疫应答。具体假设3：肝脏特异性表达将诱导对引入的GAA的耐受性并增强肌营养型AAV载体在GSD-II小鼠中的功效。对GAA的耐受性将通过免疫活性GAA-KO小鼠中GAA的肝脏特异性表达来诱导。将在耐受性GAA-KO小鼠中定量T调节细胞;此外，将耗尽T调节细胞以证明其在建立对GAA的耐受性中的作用。一种新的策略将通过静脉内注射和离体肢体递送来递送编码嵌合GAA的AAV载体，以将骨骼肌转化为分泌的GAA的贮库。GSD-II中有效的肌肉靶向基因治疗将对其他肌营养不良症和肌病的基因治疗产生影响。

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.