CN116669774A - Compositions and methods for treating brile disease - Google Patents

Abstract

Provided herein are polynucleotide sequences encoding functional human alpha-galactosidase a (hGLA) and expression cassettes containing these coding sequences. Also provided are vectors, such as recombinant adeno-associated virus (rAAV) vectors having a vector genome comprising an hGLA encoding sequence operably linked to one or more regulatory sequences. In addition, compositions containing these expression cassettes and rAAV, and methods of treating fabry disease using these compositions, are provided.

Description

Translated fromChinese

用于治疗法布里病的组合物和方法Compositions and methods for treating Fabry disease

背景技术Background Art

法布里病(Fabry disease)是一种X连锁溶酶体病症，是由负责酰基鞘鞍醇三己糖(GL-3或Gb₃)分解的酶α-半乳糖苷酶A(GLA)的基因突变引起的。GLA的缺乏导致GL-3和相关的鞘糖脂在全身血管、神经、组织和器官的血浆和细胞溶酶体中积累。这种病症是一种全身性疾病，表现为进行性肾衰竭、心脏病、脑血管疾病、小纤维周围神经病和皮肤病变等异常。导致α-半乳糖苷酶A活性缺失的GLA基因突变导致典型的、严重形式的法布里病。降低但小消除酶活性的突变通常会导致通常仅影响心脏或肾脏的更温和的迟发型法布里病。Fabry disease is an X-linked lysosomal disorder caused by mutations in the gene for alpha-galactosidase A (GLA), the enzyme responsible for the breakdown of glomerulone (GL-3 or Gb₃ ). Deficiency of GLA results in accumulation of GL-3 and related glycosphingolipids in plasma and cellular lysosomes in vessels, nerves, tissues, and organs throughout the body. The disorder is a systemic disease manifested by abnormalities such as progressive renal failure, heart disease, cerebrovascular disease, small fiber peripheral neuropathy, and skin lesions. Mutations in the GLA gene that result in loss of alpha-galactosidase A activity result in the classic, severe form of Fabry disease. Mutations that reduce but not eliminate the enzyme activity usually result in a milder, late-onset Fabry disease that usually affects only the heart or kidneys.

目前，法布里病的标准治疗包括酶替代疗法和治疗和预防疾病的其它症状的药物。在发生肾衰竭的严重病例中可能需要肾移植。Currently, standard treatment for Fabry disease includes enzyme replacement therapy and medications to treat and prevent other symptoms of the disease. In severe cases where kidney failure occurs, a kidney transplant may be required.

本领域需要用于安全有效治疗法布里病患者的组合物和方法。There is a need in the art for compositions and methods for safely and effectively treating patients with Fabry disease.

发明内容Summary of the invention

在一个方面，本文提供了包含其中包装有载体基因组的AAVhu68衣壳的重组AAV(rAAV)，其中载体基因组包含功能性人α-半乳糖苷酶A(hGLA)的编码序列和指导hGLA在靶细胞中表达的调节序列，其中编码序列包含SEQ ID NO：4的核苷酸94至1287，或与其至少85％相同的序列，并且其中hGLA在位置233和/或位置359处具有半胱氨酸残基。在某些实施例中，hGLA至少包含SEQ ID NO：2的氨基酸32至429，或与其至少95％相同的序列。在某些实施例中，hGLA包含SEQ ID NO：7的氨基酸32至429。在某些实施例中，其中hGLA包含天然信号肽。在其它实施例中，hGLA包含异源信号肽。在某些实施例中，hGLA包含SEQ ID NO：17的全长(氨基酸1至429)，或与其至少95％相同的序列。在某些实施例中，载体基因组包含组织特异性启动子。在某些实施例中，调节序列包含CB7启动子、内含子和polyA。在某些实施例中，调节序列包含土拨鼠肝炎病毒转录后调节元件(WPRE)。在某些实施例中，载体基因组包含一个或多个miRNA靶序列。In one aspect, provided herein is a recombinant AAV (rAAV) comprising an AAVhu68 capsid packaged therein with a vector genome, wherein the vector genome comprises a coding sequence for functional human α-galactosidase A (hGLA) and a regulatory sequence that directs expression of hGLA in a target cell, wherein the coding sequence comprises nucleotides 94 to 1287 of SEQ ID NO: 4, or a sequence at least 85% identical thereto, and wherein hGLA has a cysteine residue at position 233 and/or position 359. In certain embodiments, hGLA comprises at least amino acids 32 to 429 of SEQ ID NO: 2, or a sequence at least 95% identical thereto. In certain embodiments, hGLA comprises amino acids 32 to 429 of SEQ ID NO: 7. In certain embodiments, wherein hGLA comprises a native signal peptide. In other embodiments, hGLA comprises a heterologous signal peptide. In certain embodiments, hGLA comprises the full length (amino acids 1 to 429) of SEQ ID NO: 17, or a sequence at least 95% identical thereto. In certain embodiments, the vector genome comprises a tissue-specific promoter. In certain embodiments, the regulatory sequence comprises a CB7 promoter, an intron, and poly A. In certain embodiments, the regulatory sequence comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain embodiments, the vector genome comprises one or more miRNA target sequences.

在一个方面，本文提供了包含编码功能性人α-半乳糖苷酶A(hGLA)的核酸序列和指导hGLA在含有表达盒的靶细胞中表达的一个或多个调节序列的表达盒，其中核酸序列包含SEQ ID NO：4的核苷酸94至1287，或与其至少85％相同的序列，并且其中hGLA在位置233和/或位置359处具有半胱氨酸残基。在某些实施例中，hGLA包含SEQ ID NO：7的氨基酸32至429。在某些实施例中，hGLA包含天然信号肽。在其它实施例中，hGLA包含异源信号肽。在某些实施例中，hGLA包含SEQ ID NO：7的全长(氨基酸1至429)，或与其至少95％相同的序列。在某些实施例中，根据权利要求12至16中任一项所述的表达盒，其中表达盒包含组织特异性启动子。在某些实施例中，调节序列包含CB7启动子、内含子和polyA。在某些实施例中，调节序列包含土拨鼠肝炎病毒转录后调节元件(WPRE)。在某些实施例中，表达盒包含一个或多个miRNA靶序列。In one aspect, provided herein is an expression cassette comprising a nucleic acid sequence encoding functional human α-galactosidase A (hGLA) and one or more regulatory sequences that direct the expression of hGLA in a target cell containing the expression cassette, wherein the nucleic acid sequence comprises nucleotides 94 to 1287 of SEQ ID NO: 4, or a sequence at least 85% identical thereto, and wherein hGLA has a cysteine residue at position 233 and/or position 359. In certain embodiments, hGLA comprises amino acids 32 to 429 of SEQ ID NO: 7. In certain embodiments, hGLA comprises a native signal peptide. In other embodiments, hGLA comprises a heterologous signal peptide. In certain embodiments, hGLA comprises the full length (amino acids 1 to 429) of SEQ ID NO: 7, or a sequence at least 95% identical thereto. In certain embodiments, the expression cassette of any one of claims 12 to 16, wherein the expression cassette comprises a tissue-specific promoter. In certain embodiments, the regulatory sequence comprises a CB7 promoter, an intron, and polyA. In certain embodiments, the regulatory sequence comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain embodiments, the expression cassette comprises one or more miRNA target sequences.

在一个方面，本文提供了包含表达盒和一个或多个调节序列的质粒，该表达盒包含编码功能性人α-半乳糖苷酶A(hGLA)的核酸序列，该调节序列指导hGLA在含有表达盒的靶细胞中表达，其中核酸序列包含SEQ ID NO：4的核苷酸94至1287，或与其至少85％相同的序列，并且其中hGLA在位置233和/或位置359处具有半胱氨酸残基。在某些实施例中，表达盒的侧翼为AAV 5′ITR和AAV 3′ITR。在进一步的实施例中，提供了含有表达盒或质粒的宿主细胞。In one aspect, provided herein is a plasmid comprising an expression cassette comprising a nucleic acid sequence encoding functional human α-galactosidase A (hGLA) and one or more regulatory sequences that direct the expression of hGLA in a target cell containing the expression cassette, wherein the nucleic acid sequence comprises nucleotides 94 to 1287 of SEQ ID NO: 4, or a sequence at least 85% identical thereto, and wherein hGLA has a cysteine residue at position 233 and/or position 359. In certain embodiments, the expression cassette is flanked by AAV 5′ITR and AAV 3′ITR. In further embodiments, a host cell containing the expression cassette or plasmid is provided.

在又一方面，提供了包含rAAV或包含编码功能性人α-半乳糖苷酶A(hGLA)的核酸序列的表达盒的药物组合物。In yet another aspect, a pharmaceutical composition comprising rAAV or an expression cassette comprising a nucleic acid sequence encoding functional human α-galactosidase A (hGLA) is provided.

在另一方面，提供了治疗诊断为GLA缺乏症(法布里病)的人类受试者的方法，其包含向受试者施用药物组合物，该药物组合物包含rAAV或具有编码功能性人α-半乳糖苷酶A(hGLA)的序列的表达盒。在另一方面，提供了用于治疗GLA缺乏症(法布里病)的rAAV、表达盒和药物组合物。In another aspect, a method of treating a human subject diagnosed with GLA deficiency (Fabry disease) is provided, comprising administering to the subject a pharmaceutical composition comprising rAAV or an expression cassette having a sequence encoding functional human α-galactosidase A (hGLA). In another aspect, rAAV, expression cassettes, and pharmaceutical compositions for treating GLA deficiency (Fabry disease) are provided.

根据下文对本发明的详细描述，本发明的其它方面和优点将变得显而易见。Other aspects and advantages of the present invention will become apparent from the following detailed description of the invention.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1示出了CB7.CI.hGLAco(D233C_1359C).WPRE.rBG载体基因组(SEQ ID NO：6)的图谱。FIG1 shows a map of the CB7.CI.hGLAco(D233C_1359C).WPRE.rBG vector genome (SEQ ID NO: 6).

图2示出了CB7.CI.hGLAnat.WPRE.rBG载体基因组(SEQ ID NO：10)的图谱。FIG. 2 shows a map of the CB7.CI.hGLAnat.WPRE.rBG vector genome (SEQ ID NO: 10).

图3示出了TBG.PI.hGLAnat.WPRE.bGH载体基因组(SEQ ID NO：8)的图谱。FIG3 shows a map of the TBG.PI.hGLAnat.WPRE.bGH vector genome (SEQ ID NO: 8).

图4示出了CB7.CI.hGLAco.WPRE.rBG载体基因组(SEQ ID NO：14)的图谱。FIG. 4 shows a map of the CB7.CI.hGLAco.WPRE.rBG vector genome (SEQ ID NO: 14).

图5示出了TBG.PI.hGLAco.WPRE.bGH载体基因组(SEQ ID NO：12)的图谱。FIG5 shows a map of the TBG.PI.hGLAco.WPRE.bGH vector genome (SEQ ID NO: 12).

图6示出了CB7.CI.hGLAco(M51C_G360C).WPRE.rBG载体基因组(SEQ ID NO：18)的图谱。FIG. 6 shows a map of the CB7.CI.hGLAco(M51C_G360C).WPRE.rBG vector genome (SEQ ID NO: 18).

图7示出了TBG.PI.hGLAco(M51C_G360C).WPRE.bGII载体基因组(SEQ ID NO：16)的图谱。FIG. 7 shows a map of the TBG.PI.hGLAco(M51C_G360C).WPRE.bGII vector genome (SEQ ID NO: 16).

图8A和8B示出了hGLAnat(SEQ ID NO：1)、hGLAco(SEQ ID NO：3)、hGLAco(M51C_G360C)(SEQ ID NO：5)、hGLA(D233C_I359C)(SEQ ID NO：4)的核苷酸序列比对。Figures 8A and 8B show the nucleotide sequence alignment of hGLAnat (SEQ ID NO: 1), hGLAco (SEQ ID NO: 3), hGLAco(M51C_G360C) (SEQ ID NO: 5), and hGLA(D233C_I359C) (SEQ ID NO: 4).

图9示出了hGLAnat(SEQ ID NO：2)、hGLAco(SEQ ID NO：13)、hGLAco(M51C_G360C)(SEQ ID NO：17)、hGLA(D233C_I359C)(SEQ ID NO：7)的氨基酸序列比对。FIG. 9 shows the amino acid sequence alignment of hGLAnat (SEQ ID NO: 2), hGLAco (SEQ ID NO: 13), hGLAco(M51C_G360C) (SEQ ID NO: 17), and hGLA(D233C_I359C) (SEQ ID NO: 7).

图10A和图10B示出了未处理的雄性和雌性对照、Gla KO、WT/TgG3S和Gla KO/TgG3S小鼠的体重。年龄匹配的对照(WT雄性和Gla HET雌性)、Gla KO、WT/TgG3S和Gla KO/TgG3S小鼠保持未处理以评估这些模型的自然史。记录雄性(图10A)和雌性(图10B)小鼠在6周龄、12周龄、18周龄、25周龄、30周龄和35周龄的体重。给出了平均体重。误差条代表标准偏差。缩写：Gla，α-半乳糖苷酶A；TgG3S，人Gb3合酶-转基因的。Figure 10A and Figure 10B show the body weight of untreated male and female controls, Gla KO, WT/TgG3S and Gla KO/TgG3S mice. Age-matched controls (WT male and Gla HET female), Gla KO, WT/TgG3S and Gla KO/TgG3S mice remain untreated to assess the natural history of these models. The body weights of male (Figure 10A) and female (Figure 10B) mice at 6 weeks of age, 12 weeks of age, 18 weeks of age, 25 weeks of age, 30 weeks of age and 35 weeks of age were recorded. Mean body weights are given. Error bars represent standard deviations. Abbreviations: Gla, alpha-galactosidase A; TgG3S, human Gb3 synthase-transgenic.

图11A和图11B示出了未处理的雄性和雌性对照、Gla KO、WT/TgG3S和Gla KO/TgG3S小鼠的热板应答潜伏期。年龄匹配的对照(WT雄性和Gla HET雌性)、GlaKO、WT/TgG3S和Gla KO/TgG3S小鼠保持未处理以评估这些模型的自然史。在第6周、第12周、第18周、第25周、第30周和第35周时使用热板测定将每个小鼠模型中对热刺激的敏感性记录为应答潜伏期(秒)。数据表示为雄性(图11A)和雌性(图11B)小鼠在各个时间点的平均记录。误差条代表平均值的标准误差。Figure 11A and Figure 11B show the hot plate response latency of untreated male and female control, Gla KO, WT/TgG3S and Gla KO/TgG3S mice.Age-matched control (WT male and Gla HET female), GlaKO, WT/TgG3S and Gla KO/TgG3S mice remain untreated to assess the natural history of these models.At the 6th week, the 12th week, the 18th week, the 25th week, the 30th week and the 35th week, the sensitivity to thermal stimulation in each mouse model is recorded as the response latency (seconds).Data are expressed as the average record of male (Figure 11A) and female (Figure 11B) mice at each time point.Error bars represent the standard error of meansigma methods.

图12A和图12B示出了未处理的雄性和雌性对照、Gla KO、WT/TgG3S和Gla KO/TgG3S小鼠的血尿素氮(BUN)浓度。年龄匹配的对照(WT雄性和Gla HET雌性)、Gla KO、WT/TgG3S和Gla KO/TgG3S小鼠保持未处理以评估这些模型的自然史。在第6周、第12周、第18周、第25周、第30周和第35周时记录血尿素氮浓度(mg/dL)。数据表示为雄性(图12A)和雌性(图12B)小鼠在各个时间点的平均记录。误差条代表平均值的标准误差。Figure 12 A and Figure 12 B show the blood urea nitrogen (BUN) concentration of untreated male and female control, Gla KO, WT/TgG3S and Gla KO/TgG3S mice.Age-matched control (WT male and Gla HET female), Gla KO, WT/TgG3S and Gla KO/TgG3S mice remain untreated to assess the natural history of these models. Blood urea nitrogen concentration (mg/dL) was recorded at the 6th week, the 12th week, the 18th week, the 25th week, the 30th week and the 35th week. Data are expressed as the average record of male (Figure 12 A) and female (Figure 12 B) mice at each time point. Error bars represent the standard error of mean value.

图13A和图13B示出了雄性和雌性对照、Gla KO、TgG3S和Gla KO/TgG3S小鼠测量的尿渗透压。年龄匹配的对照(WT雄性和Gla HET雌性)、Gla KO、WT/TgG3S和Gla KO/TgG3S小鼠保持未处理以评估这些模型的自然史。在第25周、第30周和第35周时测量尿渗透压(mOsm/kg)。数据表示为雄性(图13A)和雌性(图13B)小鼠在各个时间点的平均记录。误差条代表平均值的标准误差。Figure 13 A and Figure 13 B show the urine osmotic pressure of male and female control, Gla KO, TgG3S and Gla KO/TgG3S mouse measurement.The control (WT male and Gla HET female) of age matching, Gla KO, WT/TgG3S and Gla KO/TgG3S mouse remain untreated to assess the natural history of these models.Urine osmotic pressure (mOsm/kg) is measured at the 25th week, the 30th week and the 35th week.Data are expressed as the average record of male (Figure 13 A) and female (Figure 13 B) mice at each time point.Error bar represents the standard error of mean value.

图14A和图14B示出了GL-3在雄性Gla KO、WT/TgG3S和Gla KO/TgG3S的肾脏中的储存。年龄匹配的对照(WT雄性和Gla HET雌性)、Gla KO、WT/TgG3S、Gla KO/TgG3S小鼠保持未处理以评估这些模型的自然史。尸检时采集肾脏和心脏，并用识别GL-3的抗体(深色沉淀)和核复染剂染色。(图14A)示出了来自雄性小鼠的代表性IHC图像。肾脏图像中的箭头表示肾小球中的储存材料。虚线圆圈区域示出了仅在一些Gla KO/TgG3S小鼠中看到的间质单核炎症(肾炎)的病灶。心脏图像中的箭头表示紧邻具有GL-3储存的心肌细胞的心肌细胞坏死和矿化。图14B是示出了使用免疫组织化学数据定量整个肾脏中GL-3储存(面积百分比)的柱状图。示出了雄性Gla KO、WT/TgG3S和Gla KO/TgG3S小鼠的结果。**p＜0.01，基于将各组与WT/TgG3S对照进行比较的克鲁斯卡尔-沃利斯检验(Kruskal-Wallis test)。Figure 14A and Figure 14B show the storage of GL-3 in the kidney of male Gla KO, WT/TgG3S and Gla KO/TgG3S.Age-matched control (WT male and Gla HET female), Gla KO, WT/TgG3S, Gla KO/TgG3S mice remain untreated to assess the natural history of these models. Kidneys and hearts were collected during autopsy and stained with antibodies (dark precipitates) and nuclear counterstains identifying GL-3. (Figure 14A) shows representative IHC images from male mice. The arrows in the kidney images represent storage materials in glomeruli. The dotted circle area shows the lesions of interstitial mononuclear inflammation (nephritis) seen only in some Gla KO/TgG3S mice. The arrows in the heart images represent myocardial cell necrosis and mineralization of myocardial cells with GL-3 storage next to each other. Figure 14B is a bar graph showing the quantitative storage of GL-3 (area percentage) in the whole kidney using immunohistochemical data. Results for male Gla KO, WT/TgG3S and Gla KO/TgG3S mice are shown. **p<0.01 based on Kruskal-Wallis test comparing each group to WT/TgG3S controls.

图15A和图15B示出了GL-3在雄性Gla KO、WT/TgG3S和Gla KO/TgG3S的背根神经节(DRG)中的储存。年龄匹配的对照(WT雄性和Gla HET雌性)、Gla KO、WT/TgG3S、Gla KO/TgG3S小鼠保持未处理以评估这些模型的自然史。尸检时采集DRG，并用识别GL-3的抗体和核复染剂染色。(图15A)示出了来自雄性小鼠的代表性图像。图15B是示出了使用免疫组织化学数据定量DRG中GL-3储存(面积百分比)的柱状图。示出了雄性Gla KO、WT/TgG3S和GlaKO/TgG3S小鼠的结果。**p＜0.01，基于将各组与WT/TgG3S对照进行比较的克鲁斯卡尔-沃利斯检验。Figures 15A and 15B show the storage of GL-3 in the dorsal root ganglia (DRG) of male Gla KO, WT/TgG3S and Gla KO/TgG3S. Age-matched controls (WT males and Gla HET females), Gla KO, WT/TgG3S, Gla KO/TgG3S mice were kept untreated to evaluate the natural history of these models. DRGs were collected at autopsy and stained with antibodies and nuclear counterstains that recognize GL-3. (Figure 15A) Representative images from male mice are shown. Figure 15B is a bar graph showing the quantitative storage of GL-3 (area percentage) in DRG using immunohistochemical data. The results of male Gla KO, WT/TgG3S and GlaKO/TgG3S mice are shown. **p<0.01, based on the Kruskal-Wallis test comparing each group to the WT/TgG3S control.

图16A至图16D示出了在Gla KO、TgG3S和Gla KO/TgG3S小鼠中通过LC-MS/MS对血浆中的lyso-Gb3和组织中的GL-3的定量。年龄匹配的对照(WT雄性和Gla HET雌性)、GlaKO、WT/TgG3S、Gla KO/TgG3S小鼠保持未处理以评估这些模型的自然史。尸检时采集肾脏、心脏和脑组织以及血浆。LC-MS/MS用于定量肾脏(图16A)、心脏(图16B)和脑组织(图16C)中的GL3或血浆中的lyso-Gb3(图16D)。对于每个图，雄性和雌性被分别绘制成图表，雌性的数据在底部。*p＜0.05，**p＜0.01克鲁斯卡尔-沃利斯检验。Figure 16 A to Figure 16 D show the quantification of lyso-Gb3 in plasma and GL-3 in tissue by LC-MS/MS in Gla KO, TgG3S and Gla KO/TgG3S mice.Age-matched control (WT male and Gla HET female), GlaKO, WT/TgG3S, Gla KO/TgG3S mice remain untreated to assess the natural history of these models. Kidney, heart and brain tissue and plasma are collected during autopsy. LC-MS/MS is used for quantitative kidney (Figure 16 A), heart (Figure 16 B) and GL3 in brain tissue (Figure 16 C) or lyso-Gb3 (Figure 16 D) in plasma. For each figure, male and female are plotted into graphs respectively, and female data are at the bottom. *p＜0.05, **p＜0.01 Kruskal-Wallis test.

图17示山了在施用对照(PBS)或三种AAVhu68.hGLA载体之一后，在第7天在Gla/小鼠的血清中测量的转基因产物表达(GLA酶活性)。以1x10¹¹GC(5.0x10¹²GC/kg)或5x10¹¹GC(2.5x10¹³GC/kg)的剂量向2至3月龄的雄性和雌性小鼠IV施用PBS(对照)或AAVhu68.hGLAnat、AAVhu68.hGLAco或AAVhu68.hGLAco(M51C_G360C)。在第1周收集血液用于血清分离，并分析GLA活性。左图示出了所有动物的汇总数据，右图和下图示出了按性别划分的结果。Figure 17 shows transgene product expression (GLA enzyme activity) measured in the serum of Gla/ mice at day 7 after administration of a control (PBS) or one of three AAVhu68.hGLA vectors. Male and female mice aged 2 to 3 months were IV administered PBS (control) or AAVhu68.hGLAnat, AAVhu68.hGLAco, or AAVhu68.hGLAco (M51C_G360C) at a dose of 1x10¹¹ GC (5.0x10¹² GC/kg) or 5x10¹¹ GC (2.5x10¹³ GC/kg). Blood was collected at week 1 for serum separation and analyzed for GLA activity. The left panel shows the aggregated data for all animals, and the right and bottom panels show the results by sex.

图18示出了施用对照(PBS)或三种AAVhu68.hGLA载体之一后，在Gla^-/-小鼠中测量的AAV基因组DNA的生物分布。以1x10¹¹GC(5.0x10¹²GC/kg)或5x10¹¹GC(2.5x10¹³GC/kg)的剂量向2至3月龄的雄性和雌性小鼠IV施用PBS(对照)或AAVhu68.hGLAnat、AAVhu68.hGLAco或AAVhu68.hGLAco(M51C_G360C)。尸检时收集肝脏样品，并分析载体分布。结果以相对于细胞基因组DNA量的转基因特异性序列的GC表示。Figure 18 shows the biodistribution of AAV genomic DNA measured in Gla^-/- mice after administration of a control (PBS) or one of three AAVhu68.hGLA vectors. PBS (control) or AAVhu68.hGLAnat, AAVhu68.hGLAco or AAVhu68.hGLAco (M51C_G360C) was administered IV to male and female mice aged 2 to 3 months at a dose of 1x10¹¹ GC (5.0x10¹² GC/kg) or 5x10¹¹ GC (2.5x10¹³ GC/kg). Liver samples were collected at autopsy and analyzed for vector distribution. Results are expressed as GC of transgene-specific sequence relative to the amount of cellular genomic DNA.

图19示出了施用三种AAVhu68.CB7.hGLA载体之一后28天，Gla KO小鼠心脏中的转基因产物表达(GLA酶活性)水平。以1x10¹¹GC(5.0x10¹²GC/kg)或5x10¹¹GC(2.5x10¹³GC/kg)的剂量向2至3月龄的雄性和雌性小鼠IV施用PBS(对照)或AAVhu68.hGLAnat(hGLA)、AAVhu68.hGLAco或AAVhu68.hGLAco(M51C_G360C)。尸检时收集心脏样品，并分析GLA活性水平。左图示出了所有动物的汇总数据，中图和右图示出了按性别划分的结果。Figure 19 shows the level of transgene product expression (GLA enzyme activity) in the heart of Gla KO mice 28 days after administration of one of the three AAVhu68.CB7.hGLA vectors. PBS (control) or AAVhu68.hGLAnat (hGLA), AAVhu68.hGLAco or^{AAVhu68.hGLAco} (M51C_G360C) were administered IV to male and female mice aged 2 to 3 months at a dose of 1x10¹¹ GC (5.0x10¹² GC/kg) or 5x10 11 GC (2.5x10¹³ GC/kg). Heart samples were collected at autopsy and analyzed for GLA activity levels. The left figure shows the summary data for all animals, and the middle and right figures show the results divided by gender.

图20示出了施用三种AAVhu68.CB7.hGLA载体之一后28天，Gla KO小鼠肝脏中的转基因产物表达(GLA酶活性)。以1x10¹¹GC(5.0x10¹²GC/kg)或5x10¹¹GC(2.5x10¹³GC/kg)的剂量向2至3月龄的雄性和雌性小鼠IV施用PBS(对照)或AAVhu68.hGLAnat(hGLA)、AAVhu68.hGLAco或AAVhu68.hGLAco(M51C_G360C)。尸检时收集肝脏样品，并分析GLA活性水平。左图示出了所有动物的汇总数据，中图和右图示出了按性别划分的结果。Figure 20 shows transgene product expression (GLA enzyme activity⁾ in the liver of Gla KO mice 28 days after administration of one of the three AAVhu68.CB7.hGLA vectors. PBS (control) or AAVhu68.hGLAnat (hGLA), AAVhu68.hGLAco or^{AAVhu68.hGLAco} (M51C_G360C) were administered IV to male and female mice aged 2 to 3 months at a dose of 1x10¹¹ GC (5.0x10¹² GC/kg) or 5x10 11 GC (2.5x10 13 GC/kg). Liver samples were collected at autopsy and analyzed for GLA activity levels. The left figure shows the summary data for all animals, and the middle and right figures show the results divided by gender.

图21示出了施用三种AAVhu68.CB7.hGLA载体之一后28天，Gla KO小鼠肾脏中的转基因产物表达(GLA酶活性)。以1x10¹¹GC(5.0x10¹²GC/kg)或5x10¹¹GC(2.5x10¹³GC/kg)的剂量向2至3月龄的雄性和雌性小鼠IV施用PBS(对照)或AAVhu68.hGLAnat(hGLA)、AAVhu68.hGLAco或AAVhu68.hGLAco(M51C_G360C)。尸检时收集肾脏样品，并分析GLA活性水平。左图示出了所有动物的汇总数据，中图和右图示出了按性别划分的结果。Figure 21 shows transgene product expression (GLA enzyme activity⁾ in the kidneys of Gla KO mice 28 days after administration of one of the three AAVhu68.CB7.hGLA vectors. PBS (control) or AAVhu68.hGLAnat (hGLA), AAVhu68.hGLAco or^{AAVhu68.hGLAco} (M51C_G360C) were administered IV to male and female mice aged 2 to 3 months at a dose of 1x10¹¹ GC (5.0x10¹² GC/kg) or 5x10 11 GC (2.5x10 13 GC/kg). Kidney samples were collected at autopsy and analyzed for GLA activity levels. The left panel shows the summary data for all animals, and the middle and right panels show the results by gender.

图22示出了施用三种AAVhu68.CB7.hGLA载体之一后28天，Gla KO小鼠脑中的转基因产物表达(GLA酶活性)。以1x10¹¹GC(5.0x10¹²GC/kg)或5x10¹¹GC(2.5x10¹³GC/kg)的剂量向2至3月龄的雄性和雌性小鼠IV施用PBS(对照)或AAVhu68.hGLAnat(hGLA)、AAVhu68.hGLAco或AAVhu68.hGLAco(M51C_G360C)。尸检时收集脑样品，并分析GLA活性水平。左图示出了所有动物的汇总数据，中图和右图示出了按性别划分的结果。Figure 22 shows transgene product expression (GLA enzyme activity⁾ in Gla KO mouse brain 28 days after administration of one of three AAVhu68.CB7.hGLA vectors. PBS (control) or AAVhu68.hGLAnat (hGLA), AAVhu68.hGLAco or AAVhu68.hGLAco (M51C_G360C) were administered IV to male and female mice aged 2 to 3 months at a dose of 1x10¹¹ GC (5.0x10¹² GC/kg) or 5x10¹¹ GC (2.5x10 13 GC/kg). Brain samples were collected at autopsy and analyzed for GLA activity levels. The left panel shows the summary data for all animals, and the middle and right panels show the results by gender.

图23示出了施用三种AAVhu68.CB7.hGLA载体之一后28天，Gla KO小鼠小肠中的转基因产物表达(GLA酶活性)。以1x10¹¹GC(5.0x10¹²GC/kg)或5x10¹¹GC(2.5x10¹³GC/kg)的剂量向2至3月龄的雄性和雌性小鼠IV施用PBS(对照)或AAVhu68.hGLAna(hGLA)、AAVhu68.hGLAco或AAVhu68.hGLAco(M51C_G360C)。尸检时收集小肠样品，并分析GLA活性水平。上图示出了所有动物的汇总数据，中图和下图示出了按性别划分的结果。Figure 23 shows transgene product expression (GLA enzyme activity) in the small intestine of Gla KO mice 28 days after administration of one of the three AAVhu68.CB7.hGLA vectors. PBS (control) or AAVhu68.hGLAna (hGLA), AAVhu68.hGLAco or^{AAVhu68.hGLAco} (M51C_G360C) were administered IV to male and female mice aged 2 to 3 months at a dose of 1x10¹¹ GC (5.0x10¹² GC/kg) or 5x10 11 GC (2.5x10¹³ GC/kg). Small intestinal samples were collected at autopsy and analyzed for GLA activity levels. The upper figure shows the summary data for all animals, and the middle and lower figures show the results divided by gender.

图24示出了施用三种AAVhu68.CB7.hGLA载体之一后，在Gla KO小鼠血浆中的lyso-Gb3储存(三己糖酰基鞘脂醇)和心脏和肾脏组织中的GL-3储存。以1x10¹¹GC(5.0x10¹²GC/kg)或5x10¹¹GC(2.5x10¹³GC/kg)的剂量向2至3月龄的雄性和雌性小鼠IV施用PBS(对照)或AAVhu68.hGLAnat(hGLA)、AAVhu68.hGLAco或AAVhu68.hGLAco(M51C_G360C)。收集血浆并通过LC-MS/MS(上图)测量储存材料lyso-Gb3的量。尸检时收集肾脏和心脏样品，并分析GL-3储存水平(分别为中图和下图)。上图示出了所有动物的汇总数据，中图和下图示出了按性别划分的结果。Figure 24 shows lyso-Gb3 storage (trihexosylceramide) in plasma of Gla KO mice and GL-3 storage in heart and kidney tissues after administration of one of three AAVhu68.CB7.hGLA vectors. PBS (control) or AAVhu68.hGLAnat (hGLA), AAVhu68.hGLAco or AAVhu68.hGLAco (M51C_G360C) were administered IV to male and female mice aged 2 to 3 months at a dose of 1x10¹¹ GC (5.0x10¹² GC/kg) or 5x10¹¹ GC (2.5x10¹³ GC/kg). Plasma was collected and the amount of storage material lyso-Gb3 was measured by LC-MS/MS (upper figure). Kidney and heart samples were collected at autopsy and analyzed for GL-3 storage levels (middle and lower figures, respectively). The upper panel shows aggregate data for all animals, the middle and lower panels show results by sex.

图25示出了施用AAV载体或媒介物后Gla KO小鼠血清中的转基因产物表达(GLA酶活性)。以2.5x10¹²GC/kg(低剂量LD)、5.0x10¹²GC/kg(中剂量；MD)，或2.5x10¹³GC/kg(高剂量；HD，仅针对AAVhu68.hGLAco(D233C_I359C))的剂量向成年(3.5至4.5月龄)雄性和雌性Gla KO或WT小鼠IV施用AAVhu68.hGLAco(WTco)、AAVhu68.hGLAco(M51C_G360C)(AT#1)或AAVhu68.hGLAco(D233C_I359C)(AT#2)。另外的Gla KO或WT小鼠IV施用媒介物(PBS)作为对照。施用后7天收集血清样品，并分析转基因产物表达(GLA酶活性)。提供了所有动物的汇总数据以及按性别划分的数据。媒介物处理的WT和Gla KO小鼠的结果是历史数据并包括在内以供参考。来自WT和Gla KO小鼠样品的历史GLA酶活性值均低于可定量的限度，因此没有绘制数据点。HD，高剂量；LD，低剂量；MD，中剂量。Figure 25 shows transgene product expression (GLA enzyme activity) in Gla^KO mouse serum after administration of AAV vector or vehicle. Adult (3.5 to 4.5 months old) male and female Gla KO or WT mice were IV administered AAVhu68.hGLAco (WTco), AAVhu68.hGLAco (M51C_G360C) (AT#1) or AAVhu68.hGLAco (D233C_I359C) (AT#2) at a dose of 2.5x10¹² GC/kg (low dose LD), 5.0x10¹² GC/kg (medium dose; MD), or 2.5x10 13 GC/kg (high dose; HD, only for AAVhu68.hGLAco (D233C_I359C)). Additional Gla KO or WT mice were IV administered vehicle (PBS) as a control. Serum samples were collected 7 days after administration and analyzed for transgenic product expression (GLA enzyme activity). Summary data for all animals and data divided by gender are provided. The results for vehicle-treated WT and Gla KO mice are historical data and are included for reference. The historical GLA enzyme activity values from WT and Gla KO mouse samples were all below the quantifiable limit, so no data points were drawn. HD, high dose; LD, low dose; MD, medium dose.

图26示出了施用AAV载体或媒介物后Gla KO小鼠血浆中的转基因产物表达(GLA酶活性)。以2.5x10¹²GC/kg(低剂量；LD)、5.0x10¹²GC/kg(中剂量；MD)，或2.5x10¹³GC/kg(高剂量；HD，仅针对AAVhu68.hGLAco(D233C_I359C))的剂量向成年(3.5至4.5月龄)雄性和雌性Gla KO或WT小鼠IV施用AAVhu68.hGLAco(WTco)、AAVhu68.hGLAco(M51C_G360C)(AT#1)或AAVhu68.hGLAco(D233C_I359C)(AT#2)。另外的Gla KO或WT小鼠IV施用媒介物(PBS)作为对照。在注射后28天收集血浆样品并分析转基因产物表达(GLA酶活性)。上图示出了所有动物的汇总数据，中图和下图示出了按性别划分的结果。Figure 26 shows transgene product expression (GLA enzyme activity) in plasma^of Gla KO mice after administration of AAV vectors or vehicles. Adult (3.5 to 4.5 months old) male and female Gla KO or WT mice were IV administered AAVhu68.hGLAco (WTco), AAVhu68.hGLAco (M51C_G360C) (AT#1) or AAVhu68.hGLAco (D233C_I359C) (AT#2) at a dose of 2.5x10¹² GC/kg (low dose; LD), 5.0x10¹² GC/kg (medium dose; MD), or 2.5x10 13 GC/kg (high dose; HD, only for AAVhu68.hGLAco (D233C_I359C)). Additional Gla KO or WT mice were IV administered vehicle (PBS) as a control. Plasma samples were collected 28 days after injection and analyzed for transgene product expression (GLA enzyme activity). The upper panel shows the aggregated data for all animals, the middle and lower panels show the results by sex.

图27示出了施用AAV载体或媒介物后Gla KO小鼠心脏中的转基因产物表达(GLA酶活性)。以2.5x10¹²GC/kg(低剂量；LD)、5.0x10¹²GC/kg(中剂量；MD)，或2.5x10¹³GC/kg(高剂量；HD，仅针对AAVhu68.hGLAco(D233C_I359C))的剂量向成年(3.5至4.5月龄)雄性和雌性Gla KO或WT小鼠IV施用AAVhu68.hGLAco(WTco)、AAVhu68.hGLAco(M51C_G360C)(AT#1)或AAVhu68.hGLAco(D233C_I359C)(AT#2)。另外的Gla KO或WT小鼠IV施用媒介物(PBS)作为对照。尸检时收集心脏样品，并分析转基因产物表达(GLA酶活性)。上图示出了所有动物的汇总数据，中图和下图示出了按性别划分的结果。媒介物处理的WT和Gla KO小鼠的结果是历史数据并包括在内以供参考。来自WT和Gla KO小鼠样品的历史GLA酶活性值均低于可定量的限度，因此没有绘制数据点。Figure 27 shows transgene product expression (GLA enzyme activity) in the heart of Gla KO mice after administration of AAV vectors or vehicles. Adult (3.5 to 4.5 months old) male^and female Gla KO or WT mice were IV administered AAVhu68.hGLAco (WTco), AAVhu68.hGLAco (M51C_G360C) (AT#1) or AAVhu68.hGLAco (D233C_I359C) (AT#2) at a dose of 2.5x10¹² GC/kg (low dose; LD), 5.0x10¹² GC/kg (medium dose; MD), or 2.5x10 13 GC/kg (high dose; HD, only for AAVhu68.hGLAco (D233C_I359C)). Additional Gla KO or WT mice were IV administered vehicle (PBS) as a control. Heart samples were collected at autopsy and transgenic product expression (GLA enzyme activity) was analyzed. The upper figure shows the summary data for all animals, and the middle and lower figures show the results divided by gender. The results of vehicle-treated WT and Gla KO mice are historical data and are included for reference. The historical GLA enzyme activity values from WT and Gla KO mouse samples are all below the limit that can be quantified, so no data points are drawn.

图28示出了施用AAV载体或媒介物后Gla KO小鼠肝脏中的转基因产物表达(GLA酶活性)。以2.5x10¹²GC/kg(低剂量；LD)、5.0x10¹²GC/kg(中剂量；MD)，或2.5x10¹³GC/kg(高剂量；HD，仅针对AAVhu68.hGLAco(D233C_I359C))的剂量向成年(3.5至4.5月龄)雄性和雌性Gla KO或WT小鼠IV施用AAVhu68.hGLAco(WTco)、AAVhu68.hGLAco(M51C_G360C)(AT#1)或AAVhu68.hGLAco(D233C_I359C)(AT#2)。另外的Gla KO或WT小鼠IV施用媒介物(PBS)作为对照。尸检时收集肝脏样品，并分析转基因产物表达(GLA酶活性)。上图示出了所有动物的汇总数据，中图和下图示出了按性别划分的结果。媒介物处理的WT和Gla KO小鼠的结果是历史数据并包括在内以供参考。来自WT和Gla KO小鼠样品的历史GLA酶活性值均低于可定量的限度，因此没有绘制数据点。Figure 28 shows transgene product expression (GLA enzyme activity) in the liver of Gla KO mice after administration of AAV vectors or vehicles. Adult (3.5 to 4.5 months old) male^and female Gla KO or WT mice were IV administered AAVhu68.hGLAco (WTco), AAVhu68.hGLAco (M51C_G360C) (AT#1) or AAVhu68.hGLAco (D233C_I359C) (AT#2) at a dose of 2.5x10¹² GC/kg (low dose; LD), 5.0x10¹² GC/kg (medium dose; MD), or 2.5x10 13 GC/kg (high dose; HD, only for AAVhu68.hGLAco (D233C_I359C)). Additional Gla KO or WT mice were IV administered vehicle (PBS) as a control. Liver samples were collected at autopsy and transgenic product expression (GLA enzyme activity) was analyzed. The upper figure shows the summary data of all animals, and the middle and lower figures show the results divided by gender. The results of vehicle-treated WT and Gla KO mice are historical data and are included for reference. The historical GLA enzyme activity values from WT and Gla KO mouse samples are all below the limit that can be quantified, so no data points are drawn.

图29示出了施用AAV载体或媒介物后Gla KO小鼠肾脏中的转基因产物表达(GLA酶活性)。以2.5x10¹²GC/kg(低剂量；LD)、5.0x10¹²GC/kg(中剂量；MD)，或2.5x10¹³GC/kg(高剂量；HD，仅针对AAVhu68.hGLAco(D233C_1359C))的剂量向成年(3.5至4.5月龄)雄性和雌性Gla KO或WT小鼠IV施用AAVhu68.hGLAco(WTco)、AAVhu68.hGLAco(M51C_G360C)(AT#1)或AAVhu68.hGLAco(D233C_I359C)(AT#2)。另外的Gla KO或WT小鼠IV施用媒介物(PBS)作为对照。尸检时收集肾脏样品，并分析转基因产物表达(GLA酶活性)。上图示山了所有动物的汇总数据，中图和下图示出了按性别划分的结果。媒介物处理的WT和Gla KO小鼠的结果是历史数据并包括在内以供参考。来自WT和Gla KO小鼠样品的历史GLA酶活性值均低于可定量的限度，因此没有绘制数据点。Figure 29 shows transgene product expression (GLA enzyme activity) in the kidney^of Gla KO mice after administration of AAV vectors or vehicles. Adult (3.5 to 4.5 months old) male and female Gla KO or WT mice were IV administered AAVhu68.hGLAco (WTco), AAVhu68.hGLAco (M51C_G360C) (AT#1) or AAVhu68.hGLAco (D233C_I359C) (AT#2) at a dose of 2.5x10¹² GC/kg (low dose; LD), 5.0x10¹² GC/kg (medium dose; MD), or 2.5x10 13 GC/kg (high dose; HD, only for AAVhu68.hGLAco (D233C_1359C)). Additional Gla KO or WT mice were IV administered vehicle (PBS) as a control. Kidney samples were collected at autopsy and analyzed for transgenic product expression (GLA enzyme activity). The upper diagram shows the summary data for all animals, and the middle and lower diagrams show the results divided by gender. The results of vehicle-treated WT and Gla KO mice are historical data and are included for reference. The historical GLA enzyme activity values from WT and Gla KO mouse samples are all below the limit that can be quantified, so no data points are drawn.

图30示出了在施用AAV载体或媒介物后收集自Gla KO小鼠的血浆中的lyso-Gb₃(三己糖酰基鞘脂醇)储存。以2.5x10¹²GC/kg(低剂量；LD)、5.0x10¹²GC/kg(中剂量；MD)，或2.5x10¹³GC/kg(高剂量；HD，仅针对AAVhu68.hGLAco(D233C_I359C))的剂量向成年(3.5至4.5月龄)雄性和雌性Gla KO或WT小鼠IV施用AAVhu68.hGLAco(WTco)、AAVhu68.hGLAco(M51C_G360C)(AT#1)或AAVhu68.hGLAco(D233C_I359C)(AT#2)。另外的Gla KO或WT小鼠IV施用媒介物(PBS)作为对照。在施用后28天进行尸检时收集血浆样品，并分析lyso-Gb₃储存水平。上图示出了所有动物的汇总数据，中图和下图示出了按性别划分的结果。媒介物处理的WT和Gla KO小鼠的结果是历史数据并包括在内以供参考。Figure 30 shows lyso-_Gb3 (trihexosylceramide) storage in plasma collected from Gla KO mice after administration of AAV vectors or vehicles. Adult (3.5 to^4.5 months old) male and female Gla KO or WT mice were IV administered AAVhu68.hGLAco (WTco), AAVhu68.hGLAco (M51C_G360C) (AT#1) or AAVhu68.hGLAco (D233C_I359C) (AT#2) at a dose of^2.5x1012 GC/kg (low dose; LD),^5.0x1012 GC/kg (medium dose; MD), or 2.5x1013 GC/kg (high dose; HD, only for AAVhu68.hGLAco (D233C_I359C)). Additional Gla KO or WT mice were IV administered vehicle (PBS) as a control. Plasma samples were collected at necropsy 28 days after administration and analyzed for lyso-Gb₃ storage levels. The upper graph shows the aggregated data for all animals, and the middle and lower graphs show the results by gender. The results for vehicle-treated WT and Gla KO mice are historical data and are included for reference.

图31A和图31B示出了在施用AAV载体或媒介物后GL-3(酰基鞘鞍醇三己糖)在GlaKO小鼠肾脏中的储存。以2.5x10¹²GC/kg(低剂量；LD)、5.0x10¹²GC/kg(中剂量；MD)，或2.5x10¹³GC/kg(高剂量；HD，仅针对AAVhu68.hGLAco(D233C_1359C))的剂量向成年(3.5至4.5月龄)雄性和雌性Gla KO或WT小鼠IV施用AAVhu68.hGLAco、AAVhu68.hGLAco(M51C_G360C)(eng#1)或AAVhu68.hGLAco(D233C_1359C)(eng#2)。另外的Gla KO或WT小鼠IV施用媒介物(PBS)作为对照。(图31A)尸检时采集肾脏，并用识别GL-3(酰基鞘鞍醇三己糖，箭头)的抗体染色。示出并标记来自雄性的代表性图像。图31B是提供了GL-3+IHC信号定量的柱状图，示出了具有GL-3+沉积物的小管的百分比。*p＜0.05，**p＜0.01，***p＜0.001，****p＜0.0001，基于克鲁斯卡尔-沃利斯检验，随后是比较各组与媒介物处理的Gla KO小鼠的事后邓恩多重比较检验。Figures 31A and 31B show the storage of GL-3 (ceramide trihexose) in the kidney of GlaKO mice after administration of AAV vectors or vehicles. Adult (^{3.5 to 4.5 months old} ) male and female GlaKO or WT mice were IV administered AAVhu68.hGLAco,^{AAVhu68.hGLAco} (M51C_G360C) (eng#1) or AAVhu68.hGLAco(D233C_1359C) (eng#2) at a dose of 2.5x10 12 GC/kg (low dose; LD), 5.0x10¹² GC/kg (medium dose; MD), or 2.5x10 13 GC/kg (high dose; HD, only for AAVhu68.hGLAco(D233C_1359C)). Additional GlaKO or WT mice were IV administered vehicle (PBS) as a control. (FIG. 31A) Kidneys were harvested at necropsy and stained with an antibody recognizing GL-3 (ceramide, arrows). Representative images from males are shown and labeled. FIG. 31B is a bar graph providing quantification of GL-3+ IHC signal, showing the percentage of tubules with GL-3+ deposits. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, based on Kruskal-Wallis test, followed by post hoc Dunn's multiple comparison test comparing each group to vehicle-treated Gla KO mice.

图32A和图32B示出了在施用AAV载体或媒介物后GL-3(酰基鞘鞍醇三己糖)在GlaKO的DRG中的储存。以2.5x10¹²GC/kg(低剂量；LD)、5.0x10¹²GC/kg(中剂量；MD)，或2.5x10¹³GC/kg(高剂量；HD，仅针对AAVhu68.hGLAco(D233C_I359Cco))的剂量向成年(3.5至4.5月龄)雄性和雌性Gla KO或WT小鼠IV施用AAVhu68.hGLAco、AAVhu68.hGLAco(M51C_G360C)(eng#1)或AAVhu68.hGLAco(D233C_I359C)(eng#2)。另外的Gla KO或WT小鼠IV施用媒介物(PBS)作为对照。(图32A)尸检时与脊髓一起采集DRG，并用识别GL-3(酰基鞘鞍醇三己糖，深色沉淀物)的抗体染色。示出并标记来自雄性的代表性图像。图32B是示出了以百分比GL-3+面积表示的量化GL-3+IHC信号的柱状图。*p＜0.05，**p＜0.01，***p＜0.001，****p＜0.0001，基于克鲁斯卡尔-沃利斯检验，随后比较各组与媒介物处理的Gla KO小鼠的事后邓恩多重比较检验。Figures 32A and 32B show the storage of GL-3 (ceramide trihexose) in DRG of GlaKO after administration of AAV vector or vehicle. Adult (3.5 to 4.5 months old) male and female GlaKO or WT mice were IV administered with AAVhu68.hGLAco,^{AAVhu68.hGLAco} (M51C_G360C) (eng#1) or AAVhu68.hGLAco(D233C_I359C) (eng#2) at a dose of 2.5x10¹² GC/kg (low dose; LD), 5.0x10¹² GC/kg (medium dose; MD), or 2.5x10 13 GC/kg (high dose; HD, only for AAVhu68.hGLAco(D233C_I359Cco)). Additional GlaKO or WT mice were IV administered with vehicle (PBS) as a control. (FIG. 32A) DRGs were harvested along with the spinal cord at necropsy and stained with an antibody recognizing GL-3 (ceramide, dark precipitate). Representative images from males are shown and labeled. FIG. 32B is a bar graph showing quantified GL-3+ IHC signal expressed as percent GL-3+ area. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, based on Kruskal-Wallis test followed by post hoc Dunn's multiple comparison test comparing each group to vehicle-treated Gla KO mice.

图33示出了来自施用AAVhu68.hGLAco(hGLAco)、AAVhu68.hGLAco(M51C_G360C)(hGLAeng#1)或AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2)的AAV处理动物的血浆中体内分泌的GLA的蛋白质印迹分析。FIG. 33 shows Western blot analysis of GLA secreted in vivo in plasma from AAV-treated animals administered AAVhu68.hGLAco (hGLAco), AAVhu68.hGLAco(M51C_G360C) (hGLAeng#1), or AAVhu68.hGLAco(D233C_I359C) (hGLAeng#2).

图34A和图34B示出了在用AAVhu68.hGLAco(D233C_I359C)处理的Gla KO雄性小鼠(图34A)和雌性小鼠(图34B)中通过免疫组织化学染色的hGLA的心脏转导和表达。用低剂量-LD(2.5x10¹²GC/kg)、中剂量-MD(5x10¹²12GC/kg)或高剂量-HD(2.5x10¹³GC/kg)的AAVhu68.hGLAco、AAVhu68.hGLAco(M51C_G360C)或AAVhu68.hGLAco(D233C_I359C)IV注射3.5至4.5月龄GLA KO法布里病小鼠。注射后4周将小鼠安乐死并收集组织。心脏用锌-福尔马林固定并用石蜡包埋。hGLA的抗体用于染色转基因表达。示出了注射AAVhu68.CB7.hGLAco(D233C_I359C)的动物的代表性图片。hGLA的暗免疫染色显示，在来自心室和心房的心肌细胞中强烈且剂量依赖性的转基因表达。Figures 34A and 34B show cardiac transduction and expression of hGLA by immunohistochemical staining in Gla KO male mice (Figure 34A) and female mice (Figure 34B) treated with AAVhu68.hGLAco (D233C_I359C). 3.5 to 4.5 month old GLA KO Fabry mice were injected IV with low dose-LD (2.5x10¹² GC/kg), medium dose-MD (5x10¹² GC/kg) or high dose-HD (2.5x10¹³ GC/kg) of AAVhu68.hGLAco, AAVhu68.hGLAco (M51C_G360C) or AAVhu68.hGLAco (D233C_I359C). Mice were euthanized 4 weeks after injection and tissues were collected. Hearts were fixed with zinc-formalin and embedded in paraffin. Antibodies to hGLA were used to stain transgene expression. Representative images of animals injected with AAVhu68.CB7.hGLAco(D233C_I359C) are shown. Dark immunostaining of hGLA showed strong and dose-dependent transgene expression in cardiomyocytes from both ventricles and atria.

图35示出了IV施用LD(2.5x10¹²GC/kg)、MD(5x10¹²GC/kg)或HD(2.5x10¹³GC/kg)的AAVhu68.hGLAco(hGLAco)、AAVhu68.hGLAco(M51C_G360C)(hGLAeng#1)或AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2)后Gla KO小鼠血浆中的抗GLA滴度。35 shows anti-GLA titers in plasma of Gla KO mice following IV administration of LD (2.5×10¹² GC/kg), MD (5×10¹² GC/kg), or HD (2.5×10¹³ GC/kg) of AAVhu68.hGLAco (hGLAco), AAVhu68.hGLAco(M51C_G360C) (hGLAeng#1), or AAVhu68.hGLAco(D233C_I359C) (hGLAeng#2).

图36A和图36B示出了单次IV剂量的AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2)后成年NHP中的AST和ALT浓度。成年NHP(N＝4)接受单次IV施用AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2)，剂量为2.5x10¹³GC/kg。在基线、第0天、第3天、第7天、第14天、第28天和第60天收集血液，并分析AST(图36A)和ALT(图36B)浓度。虚线表示参考值。缩写：ALT，丙氨酸转氨酶；AST，天冬氨酸转氨酶；GC，基因组拷贝；GGT，γ-谷氨酰转移酶。Figures 36A and 36B show AST and ALT concentrations in adult NHPs after a single IV dose of AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2). Adult NHPs (N=4) received a single IV administration of AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2) at a dose of^2.5x1013 GC/kg. Blood was collected at baseline, day 0, day 3, day 7, day 14, day 28, and day 60, and analyzed for AST (Figure 36A) and ALT (Figure 36B) concentrations. Dashed lines represent reference values. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GC, genome copies; GGT, gamma-glutamyltransferase.

图37A至图37C示出了在单次IV剂量的AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2)后成年NHP中的总胆红素(TBil)水平、血小板计数和白细胞(WBC)计数。成年NHP(N＝4)接受单次IV施用AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2)，剂量为2.5x10¹³GC/kg。在基线、第0天、第3天、第7天、第14天、第28天和第60天收集血液，并分析TBil水平(图37A)、血小板计数(图37B)和WBC计数(图37C)。虚线表示参考值。Figures 37A to 37C show total bilirubin (TBil) levels, platelet counts, and white blood cell (WBC) counts in adult NHPs after a single IV dose of AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2). Adult NHPs (N=4) received a single IV administration of AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2) at a dose of 2.5x10¹³ GC/kg. Blood was collected at baseline, day 0, day 3, day 7, day 14, day 28, and day 60, and analyzed for TBil levels (Figure 37A), platelet counts (Figure 37B), and WBC counts (Figure 37C). The dotted lines represent reference values.

图38A至图38C示出了单次Iv剂量的AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2)后成年NHP中的PT(凝血酶原时间)、APTT(活化部分凝血活酶时间)和D-二聚体水平。成年NHP(N＝4)接受单次IV施用AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2)，剂量为2.5x10¹³GC/kg。在基线、第0天、第3天、第7天、第14天、第28天和第60天收集血液，并分析PT(图38A)、APTT(图38B)和D-二聚体水平(图38C)。Figures 38A to 38C show PT (prothrombin time), APTT (activated partial thromboplastin time) and D-dimer levels in adult NHPs after a single IV dose of AAVhu68.hGLAco (D233C_I359C) (hGLAeng#2). Adult NHPs (N=4) received a single IV administration of AAVhu68.hGLAco (D233C_I359C) (hGLAeng#2) at a dose of 2.5x10¹³ GC/kg. Blood was collected at baseline, day 0, day 3, day 7, day 14, day 28 and day 60, and analyzed for PT (Figure 38A), APTT (Figure 38B) and D-dimer levels (Figure 38C).

图39示出了单次IV剂量的AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2)后成年NHP中的中和抗体和非中和结合抗体。缩写：Babs＝非中和结合抗体；F＝雌性；ID＝标识；M＝雄性；Nab＝中和抗体；NHP＝非人灵长类动物。a——值是相对发光单位(RLU)与病毒对照孔(无测试样品)相比减少50％时的血清倒数稀释度。b——值是产生比阴性对照血清高3倍的平均OD450值的最高血清稀释度的倒数。c——IgG和IgM是BAbs。Figure 39 shows neutralizing and non-neutralizing binding antibodies in adult NHPs after a single IV dose of AAVhu68.hGLAco(D233C_I359C) (hGLA eng#2). Abbreviations: Babs = non-neutralizing binding antibodies; F = female; ID = identification; M = male; Nab = neutralizing antibodies; NHP = non-human primate. a - Values are the reciprocal serum dilutions at which the relative luminescence units (RLU) were reduced by 50% compared to the virus control wells (no test sample). b - Values are the reciprocal of the highest serum dilution that produced a mean OD450 value that was 3-fold higher than the negative control serum. c - IgG and IgM are BAbs.

图40示出了在单次静脉内施用AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2)后成年NHP的血浆中的转基因产物表达(GLA酶活性)。成年NHP(n＝4)接受单次IV剂量的AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2)，剂量为2.5x10¹³GC/kg。在第7天、第14天、第28天和第60天收集血浆。测量转基因产物表达(GLA酶活性)。虚线表示基线滴度。Figure 40 shows transgene product expression (GLA enzyme activity) in plasma of adult NHPs after a single intravenous administration of AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2). Adult NHPs (n=4) received a single IV dose of AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2) at a dose of 2.5x10¹³ GC/kg. Plasma was collected on days 7, 14, 28, and 60. Transgene product expression (GLA enzyme activity) was measured. The dashed line represents the baseline titer.

图41示出了在单次静脉内施用AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2)后成年NHP血浆中针对转基因产物的抗体(抗GLA抗体)。成年NHP(n＝4)接受单次IV剂量的AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2)，剂量为2.5x10¹³GC/kg。在第7天、第14天、第28天和第60天收集血浆。测量了抗转基因产物的抗体(抗GLA抗体)。虚线表示基线酶活性。Figure 41 shows antibodies to the transgene product (anti-GLA antibodies) in adult NHP plasma after a single intravenous administration of AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2). Adult NHP (n=4) received a single IV dose of AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2) at a dose of 2.5x10¹³ GC/kg. Plasma was collected on days 7, 14, 28, and 60. Antibodies to the transgene product (anti-GLA antibodies) were measured. The dashed line represents baseline enzyme activity.

图42A和图42B示出了在单次静脉内施用AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2)后成年NHP在心脏、肝脏和肾脏中的转基因产物表达(GLA酶活性)。成年NHP(n＝4)接受单次IV剂量的AAVhu68.hGLAco(D233C_I359C)(hGLA eng#2)，剂量为2.5x10¹³GC/kg。在第60天，对动物进行尸检并收集心脏、肝脏和肾脏以测量转基因产物表达(GLA酶活性)(图42A)。来自相同物种(食蟹猴)的未处理的野生型NHP的心脏组织由BioIVT提供作为基线GLA酶活性的比较物(虚线)。基于测量值计算GLA酶活性的倍数增加(图42B)。Figures 42A and 42B show transgene product expression (GLA enzyme activity) in heart, liver and kidney of adult NHPs after a single intravenous administration of AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2). Adult NHPs (n=4) received a single IV dose of AAVhu68.hGLAco(D233C_I359C)(hGLAeng#2) at a dose of^2.5x1013 GC/kg. On day 60, animals were necropsied and heart, liver and kidney were collected to measure transgene product expression (GLA enzyme activity) (Figure 42A). Heart tissue from untreated wild-type NHPs of the same species (cynomolgus monkey) was provided by BioIVT as a comparator for baseline GLA enzyme activity (dashed line). Fold increases in GLA enzyme activity were calculated based on the measured values (Figure 42B).

图43示出了在施用AAVhu68.hGLAco(D233C-I359C)(hGLA eng#2)后来自NHP的肾脏、DRG和心脏组织中转基因的ISH和GLA表达的IHC的代表性图像。FIG. 43 shows representative images of ISH of the transgene and IHC of GLA expression in kidney, DRG, and heart tissues from NHPs following administration of AAVhu68.hGLAco(D233C-I359C) (hGLA eng#2).

图44示出了在施用AAVhu68.hGLAco(D233C-I359C)(hGLA eng#2)后来自NHP的心脏组织中转基因表达(RNAscope探针)和GLA表达的ISH的代表性图像。FIG. 44 shows representative images of ISH of transgene expression (RNAscope probe) and GLA expression in cardiac tissue from NHPs following administration of AAVhu68.hGLAco(D233C-I359C) (hGLA eng#2).

图45示山了在施用AAVhu68.hGLAco(D233C-I359C)(hGLA eng#2)后来自NHP的DRG中转基因表达(RNAscope探针)和GLA表达的ISH的代表性图像。FIG. 45 shows representative images of ISH of transgene expression (RNAscope probe) and GLA expression in DRGs from NHPs following administration of AAVhu68.hGLAco(D233C-I359C) (hGLA eng#2).

具体实施方式DETAILED DESCRIPTION

本文提供了可用于治疗法布里病和/或减轻法布里病症状的组合物。Provided herein are compositions useful for treating Fabry disease and/or alleviating the symptoms of Fabry disease.

不希望受理论束缚，包括定期输注重组人α-Gal A(rhα-Gal A)，称为酶替代疗法(ERT)，目前是具有非可适应突变的法布里病患者的主要治疗选择，而具有可适应突变的患者可受益于ERT和小分子伴侣。然而，rhα-Gal A具有低的物理稳定性、短的循环半衰期和不同疾病相关组织的可变摄取，这可能限制ERT以及依赖交叉校正的基因治疗的疗效。本文提供的组合物递送稳定的hGLA，其对于基因治疗是有效的，并且在被吸收到靶组织中之前为酶在循环中保持活性提供更大的窗口。Without wishing to be bound by theory, regular infusions of recombinant human α-Gal A (rhα-Gal A), known as enzyme replacement therapy (ERT), are currently the main treatment option for Fabry disease patients with non-adaptable mutations, while patients with adaptable mutations may benefit from ERT and small molecule chaperones. However, rhα-Gal A has low physical stability, short circulation half-life, and variable uptake by different disease-related tissues, which may limit the efficacy of ERT and gene therapies that rely on cross-correction. The compositions provided herein deliver stable hGLA, which is effective for gene therapy and provides a larger window for the enzyme to remain active in the circulation before being absorbed into the target tissue.

在某些实施例中，本文所述的组合物和方法包括核酸序列、表达盒、载体、重组病毒和用于表达功能性hGLA的其它组合物和方法。在某些实施例中，本文所述的组合物和方法包括核酸序列、表达盒、载体、重组病毒、宿主细胞、用于产生包含编码功能性hGLA或hGLA多肽的核酸序列的组合物的其它组合物和方法。在又一实施例中，本文所述的组合物和方法包括核酸序列、表达盒、载体、重组病毒、用于将编码功能性hGLA的核酸序列递送至受试者以治疗法布里病的其它组合物和方法。在一个实施例中，本文所述的组合物和方法可用于在受试者的外周(例如，血液、肝脏、肾脏)和/或外周神经系统中提供治疗水平的hGLA。在某些实施例中，本文所述的基于腺相关病毒(AAV)载体的方法提供了新的治疗选择，其通过在有需要的受试者中提供hGLA的表达来帮助恢复hGLA的所需功能和缓解与hGLA缺乏症(法布里病)相关的症状。In certain embodiments, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, and other compositions and methods for expressing functional hGLA. In certain embodiments, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, host cells, and other compositions and methods for producing compositions comprising nucleic acid sequences encoding functional hGLA or hGLA polypeptides. In yet another embodiment, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, and other compositions and methods for delivering nucleic acid sequences encoding functional hGLA to subjects to treat Fabry disease. In one embodiment, the compositions and methods described herein can be used to provide therapeutic levels of hGLA in the periphery (e.g., blood, liver, kidney) and/or peripheral nervous system of a subject. In certain embodiments, the methods described herein based on adeno-associated virus (AAV) vectors provide new treatment options that help restore the desired function of hGLA and alleviate symptoms associated with hGLA deficiency (Fabry disease) by providing expression of hGLA in subjects in need.

如本文所用，术语“治疗水平”意指酶活性为健康对照的至少约5％、约10％、约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％、约100％、大于100％、约2倍、约3倍或约5倍。用于测量hGLA酶活性的合适测定法是本领域技术人员已知的。在一些实施例中，hGLA的这种治疗水平可能导致法布里病相关症状的缓解；法布里病相关的疾病生物标志物的改善(例如，血清、尿液和/或其它生物样品中Gb3水平的降低)；促进法布里病的其它治疗(例如，酶替代或伴侣疗法)；预防神经认知衰退；逆转某些法布里病相关症状和/或预防法布里病相关症状的进展；或其任何组合。As used herein, the term "therapeutic level" means that the enzyme activity is at least about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, greater than 100%, about 2 times, about 3 times or about 5 times that of a healthy control. Suitable assays for measuring hGLA enzyme activity are known to those skilled in the art. In some embodiments, such therapeutic levels of hGLA may result in relief of symptoms associated with Fabry disease; improvement of disease biomarkers associated with Fabry disease (e.g., reduction of Gb3 levels in serum, urine and/or other biological samples); promotion of other treatments for Fabry disease (e.g., enzyme replacement or companion therapy); prevention of neurocognitive decline; reversal of certain Fabry disease-related symptoms and/or prevention of progression of Fabry disease-related symptoms; or any combination thereof.

如本文所用，“健康对照”是指受试者或来自其的生物样品，其中受试者不患有法布里病或hGLA缺乏症。健康对照可以来自一个受试者。在另一实施例中，健康对照是来自多个受试者的合并样品。As used herein, "healthy control" refers to a subject or a biological sample therefrom, wherein the subject does not suffer from Fabry disease or hGLA deficiency. A healthy control can be from one subject. In another embodiment, a healthy control is a pooled sample from multiple subjects.

如本文所用，术语“生物样品”是指任何细胞、生物流体或组织。适用于本发明的样品可以包括但不限于全血、白细胞、成纤维细胞、血清、尿液、血浆、唾液、骨髓、脑脊液、羊水和皮肤细胞。此类样品可以进一步用盐水、缓冲液或生理上可接受的稀释剂稀释。替代地，通过常规方法浓缩这些样品。As used herein, the term "biological sample" refers to any cell, biological fluid or tissue. Samples suitable for use in the present invention may include, but are not limited to, whole blood, leukocytes, fibroblasts, serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amniotic fluid and skin cells. Such samples may be further diluted with saline, buffer or a physiologically acceptable diluent. Alternatively, these samples are concentrated by conventional methods.

关于本文的描述，旨在本文描述的每种载体和其它组合物在另一实施例中是有用的。另外，在另一实施例中，本文描述的可用于所述方法的每种组合物本身也是本发明的实施例。With respect to the description herein, it is intended that each vector and other composition described herein is useful in another embodiment. Additionally, in another embodiment, each composition described herein that can be used in the methods is itself an embodiment of the present invention.

除非在本说明书中另有定义，否则本文使用的技术和科学术语具有与本发明所属领域的普通技术人员通过参考公开文本通常理解的相同含义，这些公开文本为本领域技术人员提供了对本申请中使用的许多术语的一般指导。Unless otherwise defined in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs by referring to the public texts which provide them with a general guide to many of the terms used in this application.

如本文所用，“疾病”、“病症”和“病状”是指受试者体内的法布里病和/或hGLA缺乏症。As used herein, "disease," "disorder," and "condition" refer to Fabry disease and/or hGLA deficiency in a subject.

如本文所用，术语“法布里病相关症状”或“症状”是指在法布里病患者以及法布里病动物模型中发现的症状。这些症状包括但不限于血管角质瘤、肢端感觉异常、少汗症/无汗症、角膜、晶状体混浊、心脏问题、疼痛和肾功能下降。此外，法布里病的常见心脏相关体征和症状包括左心室肥大、瓣膜病(尤其是二尖瓣脱垂和/或反流)、早发性冠状动脉疾病、心绞痛、心肌梗塞、传导异常、心律失常、充血性心力衰竭。法布里病被称为其它名称，包括α-半乳糖苷酶A缺乏症、安德森-法布里病和弥漫性体血管角质瘤。As used herein, the term "Fabry disease-related symptoms" or "symptoms" refers to symptoms found in Fabry disease patients and animal models of Fabry disease. These symptoms include, but are not limited to, angiokeratoma, acroesthesia, hypohidrosis/anhidrosis, cornea, lens opacities, cardiac problems, pain, and decreased renal function. In addition, common cardiac-related signs and symptoms of Fabry disease include left ventricular hypertrophy, valvular disease (especially mitral valve prolapse and/or regurgitation), premature coronary artery disease, angina, myocardial infarction, conduction abnormalities, arrhythmias, congestive heart failure. Fabry disease is known by other names, including α-galactosidase A deficiency, Anderson-Fabry disease, and diffuse body angiokeratoma.

本文所用的“患者”或“受试者”是指用于临床研究的男性或女性人类、狗和动物模型。在某些实施例中，这些方法和组合物的受试者是被诊断患有法布里病的人类。在进一步的实施例中，这些方法和组合物的人类受试者是产前、新生儿、婴儿、幼儿、学龄前儿童、学龄儿童、青少年、青年或成人。As used herein, "patient" or "subject" refers to male or female humans, dogs, and animal models used in clinical studies. In certain embodiments, the subject of these methods and compositions is a human diagnosed with Fabry disease. In further embodiments, the human subject of these methods and compositions is a prenatal, newborn, infant, toddler, preschooler, school-age child, adolescent, young adult, or adult.

“包含”是包括其它组分或方法步骤的术语。当使用“包含”时，应当理解，相关实施例包括使用“由......组成”术语和“基本上由......组成”术语的描述，“由......组成”术语排除了其它组分或方法步骤，“基本上由......组成”术语排除了实质上改变实施例或发明的性质的任何组分或方法步骤。应当理解，虽然本说明书中的各种实施例使用“包含”语言来呈现，但是在各种情况下，还使用“由......组成”或“基本上由......组成”语言来描述相关实施例。"Comprising" is a term that includes other components or method steps. When "comprising" is used, it should be understood that the relevant embodiments include descriptions using the terms "consisting of" and "consisting essentially of", and the "consisting of" terms exclude other components or method steps, and the "consisting essentially of" terms exclude any components or method steps that substantially change the nature of the embodiment or invention. It should be understood that although various embodiments in this specification are presented using "comprising" language, in various cases, "consisting of" or "consisting essentially of" language is also used to describe the relevant embodiments.

在描述实施例中对“一个实施例”、“另一实施例”或“某一实施例”的引用并不暗示所引用的实施例与另一实施例(例如，在所引用的实施例之前描述的实施例)相互排斥，除非另有明确规定。Reference to "one embodiment," "another embodiment," or "an embodiment" in describing an embodiment does not imply that the referenced embodiment is mutually exclusive of another embodiment (eg, an embodiment described before the referenced embodiment) unless explicitly stated otherwise.

应注意，术语“一(a)”或“一种(an)”是指一个或多个，例如，“表达盒”应理解为表示一个或多个表达盒。因此，术语“一”(或“一个”)、“一个或多个”和“至少一个”在本文中可互换使用。It should be noted that the term "a" or "an" refers to one or more, for example, "expression cassette" should be understood to mean one or more expression cassettes. Therefore, the terms "a" (or "one"), "one or more" and "at least one" are used interchangeably herein.

如本文所用，除非另有说明，否则术语“约”是指与所给出的参考相比±10％的可变性。As used herein, unless otherwise indicated, the term "about" refers to a variability of ± 10% compared to a given reference.

1.人α-半乳糖苷酶A(hGLA)1. Human α-galactosidase A (hGLA)

如本文所用，术语“人α-半乳糖苷酶A”和“hGLA”可互换使用，指人α-半乳糖苷酶A酶。α-半乳糖苷酶A的替代名称包括半乳糖苷酶α(agalsidase alfa)、α-D-半乳糖苷酶A、α-D-半乳糖苷半乳糖水解酶、α-半乳糖苷酶、α-半乳糖苷酶A、神经酰胺已三糖苷酶、GALA、半乳糖苷酶、α和蜜二糖酶。应当理解，希腊字母“alpha”和符号“α”在整个说明书中可以互换使用。包括天然(野生型)hGLA蛋白，特别是由本文提供的核酸序列表达的变体hGLA蛋白或其功能片段，当在组合物中递送或通过本文提供的方法递送时，其恢复期望的功能，缓解症状，改善与法布里病相关生物标志物(例如血清α-GAL)相关的症状，和/或促进对法布里病的其它治疗。As used herein, the terms "human α-galactosidase A" and "hGLA" are used interchangeably to refer to human α-galactosidase A enzyme. Alternative names for α-galactosidase A include agalsidase alfa, α-D-galactosidase A, α-D-galactoside galactose hydrolase, α-galactosidase, α-galactosidase A, ceramide hexatriosidase, GALA, galactosidase, α and melibiase. It should be understood that the Greek letter "alpha" and the symbol "α" are used interchangeably throughout the specification. Including natural (wild type) hGLA protein, in particular, variant hGLA protein expressed by the nucleic acid sequence provided herein or its functional fragment, when delivered in a composition or delivered by the method provided herein, it restores the desired function, alleviates symptoms, improves symptoms associated with Fabry disease-related biomarkers (e.g., serum α-GAL), and/or promotes other treatments for Fabry disease.

“人α-半乳糖苷酶A”或“hGLA”可以是例如全长蛋白质(包括信号肽和成熟蛋白)、成熟蛋白、本文所述的变体蛋白或功能片段。如本文所用，术语“功能性hGLA”是指具有全长天然(野生型)蛋白质(如SEQ ID NO：2和UniProtKB入藏号：P06280-1所示)的氨基酸序列的酶、其变体(包括本文所述的具有特定氨基酸取代的那些)、具有保守氨基酸取代的其突变体、其片段、具有保守氨基酸取代的变体和突变体的全长或仟何组合的片段，其提供至少约10％、至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约75％、至少约80％、至少约90％的天然(野生型)hGLA生物活性水平，或大约与其相同，或大于其100％。"Human α-galactosidase A" or "hGLA" can be, for example, a full-length protein (including a signal peptide and a mature protein), a mature protein, a variant protein as described herein, or a functional fragment. As used herein, the term "functional hGLA" refers to an enzyme having the amino acid sequence of a full-length native (wild-type) protein (as shown in SEQ ID NO: 2 and UniProtKB Accession No.: P06280-1), a variant thereof (including those with specific amino acid substitutions as described herein), a mutant thereof with conservative amino acid substitutions, a fragment thereof, a full-length or any combination of variants and mutants with conservative amino acid substitutions, which provides at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90% of the biological activity level of native (wild-type) hGLA, or about the same as, or greater than 100% thereof.

人α-半乳糖苷酶A-(SEQ ID NO：2)信号肽(氨基酸1至31)Human α-galactosidase A - (SEQ ID NO: 2)signal peptide (amino acids 1 to 31)

天然人GLA编码序列(SEQ ID NO：1)(参见NCBI参考序列：NM_000169.)信号肽(核苷酸1至93)Native human GLA coding sequence (SEQ ID NO: 1) (See NCBI Reference Sequence: NM_000169.)Signal peptide (nucleotides 1 to 93)

参照SEQ ID NO：2的全长天然hGLA的编号，在氨基酸位置1至31存在信号肽，成熟蛋白包括氨基酸32至429。如本文所用，“信号肽”是指存在于新合成的蛋白质的N-末端的短肽(通常约16至35个氨基酸)。信号肽，以及在一些情况下编码这种肽的核酸序列，也可以称为信号序列、靶向信号、定位信号、定位序列、转运肽、前导序列或前导肽。如本文所述，hGLA可以包括天然信号肽(即SEQ ID NO：2的氨基酸1至31)或替代地异源信号肽。在某些实施例中，hGLA是成熟蛋白(缺少信号肽序列)。With reference to the numbering of the full-length native hGLA of SEQ ID NO: 2, a signal peptide is present at amino acid positions 1 to 31, and the mature protein includes amino acids 32 to 429. As used herein, "signal peptide" refers to a short peptide (usually about 16 to 35 amino acids) present at the N-terminus of a newly synthesized protein. A signal peptide, and in some cases a nucleic acid sequence encoding such a peptide, may also be referred to as a signal sequence, a targeting signal, a localization signal, a localization sequence, a transit peptide, a leader sequence, or a leader peptide. As described herein, hGLA may include a natural signal peptide (i.e., amino acids 1 to 31 of SEQ ID NO: 2) or alternatively a heterologous signal peptide. In certain embodiments, hGLA is a mature protein (lacking a signal peptide sequence).

在某些实施例中，hGLA包括异源信号肽。在某些实施例中，这种异源信号肽优选地是人源的，并且可以包括例如IL-2信号肽。在某些实施例中可用的特定异源信号肽包括来自胰凝乳蛋白酶原B2的氨基酸1至20、人α-1-抗胰蛋白酶的信号肽、来自艾杜糖醛酸-2-硫酸酯酶的氨基酸1至25和来自蛋白酶CI抑制剂的氨基酸1至23。参见，例如，WO2018046774。其它信号/前导肽可以天然存在于免疫球蛋白(例如，IgG)、细胞因子(例如，IL-2、IL12、IL18等)、胰岛素、白蛋白、β-葡萄糖醛酸酶、碱性蛋白酶或纤连蛋白分泌信号肽等中。还参见，例如，In certain embodiments, hGLA includes a heterologous signal peptide. In certain embodiments, such a heterologous signal peptide is preferably human, and may include, for example, an IL-2 signal peptide. In certain embodiments, specific heterologous signal peptides available include amino acids 1 to 20 from chymotrypsinogen B2, a signal peptide of human α-1-antitrypsin, amino acids 1 to 25 from iduronate-2-sulfatase, and amino acids 1 to 23 from a protease CI inhibitor. See, for example, WO2018046774. Other signal/leader peptides may be naturally present in immunoglobulins (e.g., IgG), cytokines (e.g., IL-2, IL12, IL18, etc.), insulin, albumin, β-glucuronidase, alkaline protease, or fibronectin secretion signal peptides, etc. See also, for example,

signalpeptide.de/index.php？m＝listspdb_mammalia。这种嵌合hGLA可以在整个31个氨基酸的天然信号肽的位置具有异源前导序列。任选地，hGLA酶的N-末端截短可能仅缺少信号肽的一部分(例如，缺失约2至约25个氨基酸，或其间的值)、整个信号肽，或比信号肽长的片段(例如，基于SEQ ID NO：2的编号，多达70个氨基酸。任选地，这种酶可以含有长度为约5、10、15或20个氨基酸的C-末端截短。signalpeptide.de/index.php?m=listspdb_mammalia. Such chimeric hGLA may have a heterologous leader sequence in place of the entire 31 amino acid native signal peptide. Optionally, the N-terminal truncation of the hGLA enzyme may lack only a portion of the signal peptide (e.g., a deletion of about 2 to about 25 amino acids, or values therebetween), the entire signal peptide, or a fragment longer than the signal peptide (e.g., up to 70 amino acids based on numbering according to SEQ ID NO: 2. Optionally, such an enzyme may contain a C-terminal truncation of about 5, 10, 15, or 20 amino acids in length.

在某些实施例中，可以选择具有与SEQ ID NO：2的全长(氨基酸1至429)至少95％相同、至少97％相同或至少99％相同的序列的hGLA。在某些实施例中，提供了与SEQ ID NO：2的成熟蛋白(氨基酸32至429)至少95％、至少97％或至少99％相同的序列。在某些实施例中，与全长(氨基酸1至429)或成熟蛋白(氨基酸32至429)的hGLA具有至少95％至至少99％同一性的序列的特征在于当在适当的动物模型中测试时，具有与参照(即天然)hGLA相比改善的生物效应和更好的安全特性。在某些实施例中，hGLA酶在hGLA氨基酸序列的指定位置含有修饰。例如，在某些实施例中，相对于SEQ ID NO：2中的编号，hGLA在位置51和/或位置360处具有半胱氨酸取代。在某些实施例中，相对于SEQ ID NO：2中的编号，hGLA在位置233和/或位置359处具有半胱氨酸取代。此类hGLA多肽的实例提供于SEQ ID NO：7和17。In certain embodiments, hGLA having a sequence that is at least 95% identical, at least 97% identical, or at least 99% identical to the full length (amino acids 1 to 429) of SEQ ID NO: 2 can be selected. In certain embodiments, a sequence that is at least 95%, at least 97%, or at least 99% identical to the mature protein (amino acids 32 to 429) of SEQ ID NO: 2 is provided. In certain embodiments, a sequence that is at least 95% to at least 99% identical to the full length (amino acids 1 to 429) or mature protein (amino acids 32 to 429) of hGLA is characterized by having improved biological effects and better safety characteristics compared to reference (i.e., native) hGLA when tested in an appropriate animal model. In certain embodiments, the hGLA enzyme contains modifications at specified positions of the hGLA amino acid sequence. For example, in certain embodiments, hGLA has a cysteine substitution at position 51 and/or position 360 relative to the numbering in SEQ ID NO: 2. In certain embodiments, hGLA has a cysteine substitution at position 233 and/or position 359 relative to the numbering in SEQ ID NO: 2. Examples of such hGLA polypeptides are provided in SEQ ID NOs: 7 and 17.

如本文所用，“保守性氨基酸置换”或“保守性氨基酸取代”是指将氨基酸改变、置换或取代成具有类似生化特性(例如电荷、疏水性和大小)的不同氨基酸，这是本领域技术人员已知的。还参见，例如FRENCH等人《什么是保守性取代？(What is a conservativesubstitution？)》《分子进化杂志(Journal of Molecular Evolution)》，1983年3月，第19卷，第2期，第171至175页和YAMPOLSKY等人《蛋白质中氨基酸的可交换性(TheExchangeability of Amino Acids in Proteins)》，《遗传学(Genetics.)》2005年8月；170(4)：1459-1472，其各自通过全文引用的方式并入本文。As used herein, "conservative amino acid substitution" or "conservative amino acid substitution" refers to changing, replacing or substituting an amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size), which is known to those skilled in the art. See also, for example, FRENCH et al., What is a conservative substitution? Journal of Molecular Evolution, March 1983, Vol. 19, No. 2, pp. 171-175 and YAMPOLSKY et al., The Exchangeability of Amino Acids in Proteins, Genetics. August 2005; 170(4): 1459-1472, each of which is incorporated herein by reference in its entirety.

在一个方面，本文提供了编码功能性hGLA蛋白的核酸序列，以及例如包含其的表达盒和载体。在一个实施例中，核酸序列是SEQ ID NO：1中再现的野生型编码序列。在进一步的实施例中，核酸序列与SEQ ID NO：1的野生型hGLA序列至少约60％、至少约65％、至少约70％、至少约75％或至少约80％相同，并编码功能性hGLA。In one aspect, provided herein are nucleic acid sequences encoding functional hGLA proteins, and, for example, expression cassettes and vectors comprising the same. In one embodiment, the nucleic acid sequence is the wild-type coding sequence reproduced in SEQ ID NO: 1. In further embodiments, the nucleic acid sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% identical to the wild-type hGLA sequence of SEQ ID NO: 1, and encodes functional hGLA.

如本文所用，“核酸”是指核苷酸的聚合形式，包括RNA、mRNA、cDNA、基因组DNA、肽核酸(PNA)和上述的合成形式和混合聚合物。核苷酸是指核糖核苷酸、脱氧核苷酸或任一类型核苷酸的修饰形式(例如，肽核酸寡聚物)。该术语还包括单链和双链形式的DNA。本领域技术人员将理解，本文描述了这些核酸分子的功能性变体。功能性变体是可以使用标准遗传密码直接翻译以提供与从亲本核酸分子翻译的氨基酸序列相同的氨基酸序列的核酸序列。As used herein, "nucleic acid" refers to a polymeric form of nucleotides, including RNA, mRNA, cDNA, genomic DNA, peptide nucleic acids (PNA) and the above-mentioned synthetic forms and mixed polymers. Nucleotide refers to a modified form of ribonucleotides, deoxynucleotides or any type of nucleotides (e.g., peptide nucleic acid oligomers). The term also includes DNA in single-stranded and double-stranded forms. Those skilled in the art will appreciate that functional variants of these nucleic acid molecules are described herein. Functional variants are nucleic acid sequences that can be directly translated using the standard genetic code to provide an amino acid sequence identical to the amino acid sequence translated from the parent nucleic acid molecule.

在某些实施例中，编码功能性hGLA的核酸分子和本文所述的其它构建体可用于生成表达盒和载体基因组，并且可以被工程化以在酵母细胞、昆虫细胞或哺乳动物细胞(如人类细胞)中表达。方法是已知的并且先前已经描述(例如WO 96/09378)。如果与野生型序列相比，至少一个非优选密码子被更优选的密码子替代，则认为该序列是工程化的。在本文中，非优选密码子是在生物体中使用的频率低于编码相同氨基酸的另一密码子的密码子，并且更优选的密码子是在生物体中使用的频率高于非优选密码子的密码子。特定生物体的密码子使用频率可以在密码子频率表中找到，如在www.kazusa.jp/codon中。优选地多于一个非优选密码子，优选地大多数或所有非优选密码子被更优选的密码子替代。优选地，生物体中最常用的密码子用于工程化序列中。优选密码子的置换通常导致更高的表达。本领域技术人员还将理解，由于遗传密码的简并性，许多不同的核酸分子可以编码相同的多肽。还应当理解，技术人员可以使用常规技术进行不影响由核酸分子编码的氨基酸序列的核苷酸取代，以反映其中表达多肽的任何特定宿主生物体的密码子使用。因此，除非另有说明，“编码氨基酸序列的核酸序列”包括彼此为简并形式并编码相同氨基酸序列的所有核苷酸序列。核酸序列可以使用常规分子生物学技术克隆，或通过DNA合成从头生成，其可以由在DNA合成和/或分子克隆领域具有业务的服务公司(例如GeneArt、金斯瑞公司(GenScript)、生命科技公司(Life Technologies)、欧陆集团(Eurofins))使用常规程序进行。In certain embodiments, nucleic acid molecules encoding functional hGLA and other constructs described herein can be used to generate expression cassettes and vector genomes, and can be engineered to be expressed in yeast cells, insect cells or mammalian cells (such as human cells). Methods are known and have been described previously (e.g., WO 96/09378). If at least one non-preferred codon is replaced by a more preferred codon compared to the wild-type sequence, the sequence is considered to be engineered. In this article, a non-preferred codon is a codon that is used less frequently in an organism than another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage of a particular organism can be found in a codon frequency table, such as at www.kazusa.jp/codon. Preferably more than one non-preferred codon, preferably most or all non-preferred codons are replaced by more preferred codons. Preferably, the most commonly used codons in an organism are used in an engineered sequence. The replacement of preferred codons generally results in higher expression. It will also be understood by those skilled in the art that, due to the degeneracy of the genetic code, many different nucleic acid molecules can encode the same polypeptide. It should also be understood that the technician can use conventional techniques to carry out nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acid molecule to reflect the codon usage of any specific host organism in which the polypeptide is expressed. Therefore, unless otherwise indicated, "nucleic acid sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms and encode the same amino acid sequence. Nucleic acid sequences can be cloned using conventional molecular biological techniques, or generated de novo by DNA synthesis, which can be performed by service companies (e.g., GeneArt, GenScript, Life Technologies, Eurofins) with business in the field of DNA synthesis and/or molecular cloning using conventional procedures.

在某些实施例中，本文所述的核酸、表达盒、载体基因组包括作为工程化序列的hGLA编码序列。在某些实施例中，工程化序列可用于改善受试者中的生产、转录、表达或安全性。在某些实施例中，工程化序列可用于增加所得治疗组合物或治疗的疗效。在进一步的实施例中，工程化序列可用于增加所表达的功能性hGLA蛋白的疗效，并且还可以允许递送功能性hGLA的治疗试剂的较低剂量。在某些实施例中，工程化hGLA编码序列的特征在于与野生型hGLA编码序列相比提高的翻译速率。In certain embodiments, the nucleic acids, expression cassettes, vector genomes described herein include an hGLA coding sequence as an engineered sequence. In certain embodiments, the engineered sequence can be used to improve production, transcription, expression, or safety in a subject. In certain embodiments, the engineered sequence can be used to increase the therapeutic efficacy of the resulting therapeutic composition or treatment. In further embodiments, the engineered sequence can be used to increase the therapeutic efficacy of the expressed functional hGLA protein, and can also allow for lower doses of therapeutic agents that deliver functional hGLA. In certain embodiments, the engineered hGLA coding sequence is characterized by an increased translation rate compared to the wild-type hGLA coding sequence.

“工程化”是指将编码本文所述的功能性hGLA酶的核酸序列组装并置于任何合适的遗传元件中，例如裸DNA、噬菌体、转座子、粘粒、附加体等，其将其上携带的hGLA序列转移至宿主细胞，例如用于生成非病毒递送系统(例如，基于RNA的系统、裸DNA等)，或用于在包装宿主细胞中生成病毒载体，和/或用于递送至受试者体内的宿主细胞。在某些实施例中，遗传元件是载体。在一个实施例中，遗传元件是质粒。用于制备这种工程化构建体的方法是核酸操作领域技术人员已知的，并且包括基因工程、重组工程和合成技术。参见，例如，Green和Sambrook，《分子克隆：实验室手册(Molecular Cloning：A Laboratory Manual)》，冷泉港出版社(Cold Spring Harbor Press)，纽约冷泉港(2012)。"Engineering" refers to assembling and placing a nucleic acid sequence encoding a functional hGLA enzyme described herein into any suitable genetic element, such as naked DNA, bacteriophage, transposon, cosmid, episome, etc., which transfers the hGLA sequence carried thereon to a host cell, for example, for generating a non-viral delivery system (e.g., an RNA-based system, naked DNA, etc.), or for generating a viral vector in a packaging host cell, and/or for delivery to a host cell in a subject. In certain embodiments, the genetic element is a vector. In one embodiment, the genetic element is a plasmid. Methods for preparing such engineered constructs are known to those skilled in the art of nucleic acid manipulation, and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (2012).

在核酸序列的上下文中，术语“同一性百分比(％)”、“序列同一性”、“序列同一性百分比”或“同一性百分比”是指当进行对应比对时两个序列中相同的残基。序列同一性比较的长度可以超过构建体的全长、基因编码序列的全长，或至少约500至1000个核苷酸的片段。然而，也可能需要较小片段之间的同一性，例如至少约九个核苷酸，通常至少约20至24个核苷酸、至少约28至32个核苷酸、至少约36个或更多个核苷酸。In the context of nucleic acid sequences, the terms "percent identity (%)", "sequence identity", "percent sequence identity" or "percent identity" refer to the residues that are identical in two sequences when the corresponding comparison is performed. The length of the sequence identity comparison can exceed the full length of the construct, the full length of the gene coding sequence, or a fragment of at least about 500 to 1000 nucleotides. However, identity between smaller fragments may also be desired, such as at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides.

可以容易地确定蛋白质、多肽、约100个氨基酸、约300个氨基酸或其肽片段或相应的核酸序列编码序列全长上的氨基酸序列的同一性百分比。合适的氨基酸片段的长度可以是至少约8个氨基酸，并且可以高达约50个氨基酸。通常，当提及两个不同序列之间的“同一性”、“同源性”或“相似性”时，“同一性”、“同源性”或“相似性”是参照“比对的”序列确定的。“比对”序列或“比对”是指多个核酸序列或蛋白质(氨基酸)序列，与参考序列相比，通常含有缺失或额外碱基或氨基酸的校正。The percent identity of an amino acid sequence over the full length of a protein, a polypeptide, about 100 amino acids, about 300 amino acids, or a peptide fragment thereof, or a corresponding nucleic acid sequence encoding sequence can be readily determined. Suitable amino acid fragments can be at least about 8 amino acids in length, and can be up to about 50 amino acids. Typically, when referring to "identity," "homology," or "similarity" between two different sequences, the "identity," "homology," or "similarity" is determined with reference to an "aligned" sequence. An "aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences, typically containing corrections for missing or extra bases or amino acids compared to a reference sequence.

同一性可以通过制备序列的比对并通过使用本领域已知或商业上可获得的各种算法和/或计算机程序(例如，BLAST，ExPASy；Clustal Omega；FASTA；使用例如尼德勒曼-翁施算法、史密斯-沃特曼算法)确定。使用各种公开的或商业上可获得的多序列比对程序中的任一种进行比对。序列比对程序可用于氨基酸序列，例如“Clustal Omega”、“ClustalX”、“MAP”、“PIMA”、“MSA”、“BLOCKMAKER”、“MEME”和“Match-Box”程序。通常，在默认设置下使用这些程序中的任何一个，但是本领域技术人员可以根据需要改变这些设置。替代地，本领域技术人员可以利用另一种算法或计算机程序，其至少提供由所引用的算法和程序提供的同一性或比对水平。参见，例如，J.D.Thomson等人，《核酸研究(Nucl.Acids.Res.)》，《多序列比对的综合比较(A comprehensive comparison of multiple sequencealignments)》，27(13)：2682-2690(1999)。Identity can be determined by preparing a comparison of the sequence and by using various algorithms and/or computer programs known in the art or commercially available (e.g., BLAST, ExPASy; Clustal Omega; FASTA; using, for example, the Needleman-Wunsch algorithm, the Smith-Waterman algorithm). The comparison is performed using any of the various public or commercially available multiple sequence alignment programs. Sequence alignment programs can be used for amino acid sequences, such as "Clustal Omega", "ClustalX", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used under the default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize another algorithm or computer program that at least provides the identity or alignment level provided by the cited algorithm and program. See, e.g., J.D. Thomson et al., Nucl. Acids. Res., A comprehensive comparison of multiple sequence alignments, 27(13): 2682-2690 (1999).

在某些实施例中，hGLA编码序列与SEQ ID NO：1的野生型hGLA序列的同一性小于80％，并且编码SEQ ID NO：2、7或17的氨基酸序列。在另一实施例中，hGLA编码序列包含与SEQ ID NO：1的核苷酸(nt)94至1287小于80％相同的序列，并且编码SEQ ID NO：2、7或17的氨基酸32至429。In certain embodiments, the hGLA coding sequence is less than 80% identical to the wild-type hGLA sequence of SEQ ID NO: 1, and encodes the amino acid sequence of SEQ ID NO: 2, 7, or 17. In another embodiment, the hGLA coding sequence comprises a sequence less than 80% identical to nucleotides (nt) 94 to 1287 of SEQ ID NO: 1, and encodes amino acids 32 to 429 of SEQ ID NO: 2, 7, or 17.

在某些实施例中，hGLA编码序列与野生型hGLA编码序列(SEQ ID NO：1)共有小于约99％、小于约98％、小于约97％、小于约96％、小于约95％、小于约94％、小于约93％、小于约92％、小于约91％、小于约90％、小于约89％、小于约88％、小于约87％、小于约86％、小于约85％、小于约84％、小于约83％、小于约82％、小于约81％、小于约80％、小于约79％、小于约78％、小于约77％、小于约76％、小于约75％、小于约74％、小于约73％、小于约72％、小于约71％、小于约70％、小于约69％、小于约68％、小于约67％、小于约66％、小于约65％、小于约64％、小于约63％、小于约62％、小于约61％或同一性。在其它实施例中，hGLA编码序列与野生型hGLA编码序列(SEQ ID NO：1)共有约99％、约98％、约97％、约96％、约95％、约94％、约93％、约92％、约91％、约90％、约89％、约88％、约87％、约86％、约85％、约84％、约83％、约82％、约81％、约80％、约79％、约78％、约77％、约76％、约75％、约74％、约73％、约72％、约71％、约70％、约69％、约68％、约67％、约66％、约64％、约63％、约62％、约61％或更少的同一性。在另一实施例中，hGLA编码序列与SEQ ID NO：3至少约80％、至少约81％、至少约82％、至少约83％、至少约84％、至少约85％、至少约86％、至少约87％、至少约88％、至少约89％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％相同，并且该序列编码功能性hGLA。同一性可以是相对于编码全长hGLA的序列(例如，SEQ ID NO：1或3的nt 1至nt 1287)或相对于编码成熟hGLA的序列(例如，SEQ ID NO：1或3的nt 94至nt 1287)。在某些实施例中，hGLA编码序列包括SEQ ID NO：3的nt 1至1287，或与其至少85％、90％、95％或99％相同的编码全长hGLA的序列。在某些实施例中，hGLA编码序列包括SEQ ID NO：3的nt 94至1287，或与其至少85％、90％、95％或99％相同的编码功能性hGLA的序列。In certain embodiments, the hGLA coding sequence is identical to the wild-type hGLA coding sequence (SEQ ID NO: 1) shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61% or identity. In other embodiments, the hGLA coding sequence shares about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about 70%, about 69%, about 68%, about 67%, about 66%, about 64%, about 63%, about 62%, about 61% or less identity with the wild-type hGLA coding sequence (SEQ ID NO: 1). In another embodiment, the hGLA coding sequence is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to SEQ ID NO: 3, and the sequence encodes functional hGLA. The identity can be relative to the sequence encoding full-length hGLA (e.g., nt 1 to nt 1287 of SEQ ID NO: 1 or 3) or relative to the sequence encoding mature hGLA (e.g., nt 94 to nt 1287 of SEQ ID NO: 1 or 3). In certain embodiments, the hGLA coding sequence comprises nt 1 to 1287 of SEQ ID NO: 3, or a sequence encoding full-length hGLA that is at least 85%, 90%, 95% or 99% identical thereto. In certain embodiments, the hGLA coding sequence comprises nt 94 to 1287 of SEQ ID NO: 3, or a sequence encoding functional hGLA that is at least 85%, 90%, 95% or 99% identical thereto.

在某些实施例中，提供了在位置233和/或位置359处具有氨基取代的hGLA，其中参考SEQ ID NO：2的全长天然hGLA的编号。在某些实施例中，hGLA在位置233和/或位置359处具有半胱氨酸残基。在某些实施例中，hGLA包含SEQ ID NO：7的氨基酸序列，或与其至少95％相同的在位置233和位置359处具有半胱氨酸残基的序列。在其它实施例中，hGLA包含SEQ ID NO：7的氨基酸32至429，或与其至少95％相同的在位置233和位置359处具有半胱氨酸残基的序列。在某些实施例中，提供了编码SEQ ID NO：7的序列的工程化编码序列，或与其至少95％相同的在位置233和位置359处具有半胱氨酸残基的序列，其中该编码序列与野生型hGLA编码序列(SEQ ID NO：1)共有小于约99％、小于约98％、小于约97％、小于约96％、小于约95％、小于约94％、小于约93％、小于约92％、小于约91％、小于约90％、小于约89％、小于约88％、小于约87％、小于约86％、小于约85％、小于约84％、小于约83％、小于约82％、小于约81％、小于约80％、小于约79％、小于约78％、小于约77％、小于约76％、小于约75％、小于约74％、小于约73％、小于约72％、小于约71％、小于约70％、小于约69％、小于约68％、小于约67％、小于约66％、小于约65％、小于约64％、小于约63％、小于约62％、小于约61％或同一性。在其它实施例中，提供了编码SEQ ID NO：7的氨基酸32至429的工程化编码序列，或与其至少95％相同的在位置233和位置359处具有半胱氨酸残基的序列，其中该编码序列与成熟hGLA的野生型编码序列(SEQ ID NO：1的nt 94至nt1287)共有小于约99％、小于约98％、小于约97％、小于约96％、小于约95％、小于约94％、小于约93％、小于约92％、小于约91％、小于约90％、小于约89％、小于约88％、小于约87％、小于约86％、小于约85％、小于约84％、小于约83％、小于约82％、小于约81％、小于约80％、小于约79％、小于约78％、小于约77％、小于约76％、小于约75％、小于约74％、小于约73％、小于约72％、小于约71％、小于约70％、小于约69％、小于约68％、小于约67％、小于约66％、小于约65％、小于约64％、小于约63％、小于约62％、小于约61％或同一性。在某些实施例中，提供了hGLA编码序列，其包含SEQ ID NO：4的nt 94至nt 1287，或与其至少85％、90％、95％或99％相同的序列，其中编码的功能性hGLA在位置233和位置359处具有半胱氨酸残基。在某些实施例中，hGLA编码序列包含SEQ ID NO：4的nt 94至1287。在另一实施例中，提供了hGLA编码序列，其包含SEQ IDNO：4，或与其至少85％、90％、95％或99％相同的序列，其中编码的功能性hGLA在位置233和位置359处具有半胱氨酸残基。在某些实施例中，hGLA编码序列包含SEQ ID NO：4。In certain embodiments, hGLA is provided that has an amino substitution at position 233 and/or position 359, with reference to the numbering of the full-length native hGLA of SEQ ID NO: 2. In certain embodiments, hGLA has a cysteine residue at position 233 and/or position 359. In certain embodiments, hGLA comprises the amino acid sequence of SEQ ID NO: 7, or a sequence at least 95% identical thereto having cysteine residues at positions 233 and 359. In other embodiments, hGLA comprises amino acids 32 to 429 of SEQ ID NO: 7, or a sequence at least 95% identical thereto having cysteine residues at positions 233 and 359. In certain embodiments, an engineered coding sequence encoding a sequence of SEQ ID NO: 7, or a sequence at least 95% identical thereto having cysteine residues at positions 233 and 359, is provided, wherein the coding sequence is identical to the wild-type hGLA coding sequence (SEQ ID NO: 1) shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61% or identity. In other embodiments, an engineered coding sequence encoding amino acids 32 to 429 of SEQ ID NO:7, or a sequence at least 95% identical thereto having cysteine residues at position 233 and position 359, wherein the coding sequence shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81% with the wild-type coding sequence of mature hGLA (nt 94 to nt 1287 of SEQ ID NO:1). , less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61%, or identity. In certain embodiments, an hGLA coding sequence is provided that comprises nt 94 to nt 1287 of SEQ ID NO: 4, or a sequence that is at least 85%, 90%, 95%, or 99% identical thereto, wherein the encoded functional hGLA has cysteine residues at positions 233 and 359. In certain embodiments, the hGLA coding sequence comprises nt 94 to 1287 of SEQ ID NO: 4. In another embodiment, a hGLA coding sequence is provided, comprising SEQ ID NO: 4, or a sequence at least 85%, 90%, 95% or 99% identical thereto, wherein the encoded functional hGLA has cysteine residues at position 233 and position 359. In certain embodiments, the hGLA coding sequence comprises SEQ ID NO: 4.

在某些实施例中，提供了在位置51和/或位置360处具有氨基取代的hGLA，其中参考SEQ ID NO：2)的全长天然hGLA的编号。在某些实施例中，hGLA在位置51和/或位置360处具有半胱氨酸残基。在某些实施例中，hGLA包含SEQ ID NO：17的氨基酸序列，或与其至少95％相同的在位置51和位置360处具有半胱氨酸残基的序列。在其它实施例中，hGLA包含SEQ ID NO：17的氨基酸32至429，或与其至少95％相同的在位置51和位置360处具有半胱氨酸残基的序列。在某些实施例中，提供了编码SEQ ID NO：17的序列的工程化编码序列，或与其至少95％相同的在位置51和位置360处具有半胱氨酸残基的序列，其中该序列与野生型hGLA编码序列(SEQ ID NO：1)共有小于约99％、小于约98％、小于约97％、小于约96％、小于约95％、小于约94％、小于约93％、小于约92％、小于约91％、小于约90％、小于约89％、小于约88％、小于约87％、小于约86％、小于约85％、小于约84％、小于约83％、小于约82％、小于约81％、小于约80％、小于约79％、小于约78％、小于约77％、小于约76％、小于约75％、小于约74％、小于约73％、小于约72％、小于约71％、小于约70％、小于约69％、小于约68％、小于约67％、小于约66％、小于约65％、小于约64％、小于约63％、小于约62％、小于约61％或同一性。在其它实施例中，提供了编码SEQ ID NO：17的氨基酸32至429的工程化编码序列，或与其至少95％相同的在位置51和位置360处具有半胱氨酸残基的序列，其中该序列与成熟hGLA的野生型编码序列(SEQ ID NO：1的94至nt 1287)共有小于约99％、小于约98％、小于约97％、小于约96％、小于约95％、小于约94％、小于约93％、小于约92％、小于约91％、小于约90％、小于约89％、小于约88％、小于约87％、小于约86％、小于约85％、小于约84％、小于约83％、小于约82％、小于约81％、小于约80％、小于约79％、小于约78％、小于约77％、小于约76％、小于约75％、小于约74％、小于约73％、小于约72％、小于约71％、小于约70％、小于约69％、小于约68％、小于约67％、小于约66％、小于约65％、小于约64％、小于约63％、小于约62％、小于约61％或同一性。在某些实施例中，提供了hGLA编码序列，其包含SEQ ID NO：5的nt 94至nt 1287，或与其至少85％、90％、95％或99％相同的序列，其中编码的功能性hGLA在位置51和位置360处具有半胱氨酸残基。在某些实施例中，hGLA编码序列包含SEQ IDNO：5的nt 94至nt 1287。在另一实施例中，提供了hGLA编码序列，其包含SEQ ID NO：5，或与其至少85％、90％、95％或99％相同的序列，其中编码的功能性hGLA在位置51和位置360处具有半胱氨酸残基。在某些实施例中，hGLA编码序列包含SEQ ID NO：5。In certain embodiments, hGLA is provided that has an amino substitution at position 51 and/or position 360, with reference to the numbering of the full-length native hGLA of SEQ ID NO: 2). In certain embodiments, hGLA has a cysteine residue at position 51 and/or position 360. In certain embodiments, hGLA comprises the amino acid sequence of SEQ ID NO: 17, or a sequence at least 95% identical thereto having cysteine residues at positions 51 and 360. In other embodiments, hGLA comprises amino acids 32 to 429 of SEQ ID NO: 17, or a sequence at least 95% identical thereto having cysteine residues at positions 51 and 360. In certain embodiments, an engineered coding sequence encoding a sequence of SEQ ID NO: 17, or a sequence at least 95% identical thereto having cysteine residues at positions 51 and 360, is provided, wherein the sequence is identical to the wild-type hGLA coding sequence (SEQ ID NO: 1) shares less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61% or identity. In other embodiments, an engineered coding sequence encoding amino acids 32 to 429 of SEQ ID NO: 17, or a sequence at least 95% identical thereto having cysteine residues at positions 51 and 360, wherein the sequence is identical to the wild-type coding sequence of mature hGLA (94 to nt 1 of SEQ ID NO: 1). 1287) share less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61% or identity. In certain embodiments, a hGLA coding sequence is provided that comprises nt 94 to nt 1287 of SEQ ID NO: 5, or a sequence at least 85%, 90%, 95% or 99% identical thereto, wherein the encoded functional hGLA has cysteine residues at position 51 and position 360. In certain embodiments, the hGLA coding sequence comprises nt 94 to nt 1287 of SEQ ID NO: 5. In another embodiment, a hGLA coding sequence is provided that comprises SEQ ID NO: 5, or a sequence at least 85%, 90%, 95% or 99% identical thereto, wherein the encoded functional hGLA has cysteine residues at position 51 and position 360. In certain embodiments, the hGLA coding sequence comprises SEQ ID NO: 5.

如本文所用，“所需功能”是指hGLA酶活性为健康对照的至少约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％或约100％。As used herein, "desired function" refers to hGLA enzyme activity that is at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of that of a healthy control.

如本文所用，短语“缓解症状”和“改善症状”及其语法变体是指逆转法布里病相关症状、减缓或预防法布里病相关症状的进展。在某些实施例中，缓解或改善是指在施用所述组合物或使用所述方法后，患者的症状总数与施用或使用前相比减少了约5％、约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约95％。在另一实施例中，缓解或改善是指在施用所述组合物或使用所述方法后，症状的严重程度或进展与施用或使用前相比降低了约5％、约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约95％。As used herein, the phrases "relieve symptoms" and "improve symptoms" and grammatical variants thereof refer to reversing symptoms associated with Fabry disease, slowing down or preventing the progression of symptoms associated with Fabry disease. In certain embodiments, relief or improvement refers to that after administering the composition or using the method, the total number of symptoms of the patient is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to before administration or use. In another embodiment, relief or improvement refers to that after administering the composition or using the method, the severity or progression of the symptoms is reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% compared to before administration or use.

其它hGLA变体也是合适的。另参见2019年10月10日提交的国际专利申请号PCT/US2019/05567，其全部内容通过引用并入本文。Other hGLA variants are also suitable. See also International Patent Application No. PCT/US2019/05567, filed October 10, 2019, the entire contents of which are incorporated herein by reference.

应当理解，本文所述的功能性hGLA或hGLA编码序列中的组合物旨在应用于说明书中所述的其它组合物、方案、方面、实施例和方法。It is to be understood that the compositions of functional hGLA or hGLA encoding sequences described herein are intended to be applicable to other compositions, schemes, aspects, embodiments and methods described in the specification.

2.表达盒2. Expression Cassette

在某些实施例中，本文提供了具有编码功能性hGLA的工程化核酸序列和指导其表达的调节序列的表达盒。在其它实施例中，表达盒具有编码功能性hGLA的本文所述工程化核酸序列和指导其表达的调节序列。In certain embodiments, provided herein are expression cassettes having an engineered nucleic acid sequence encoding functional hGLA and regulatory sequences directing its expression. In other embodiments, expression cassettes have an engineered nucleic acid sequence described herein encoding functional hGLA and regulatory sequences directing its expression.

如本文所用，术语“表达”或“基因表达”是指将来自基因的信息用于合成功能性基因产物的过程。基因产物可以是蛋白质、肽或核酸聚合物(如RNA、DNA或PNA)。As used herein, the term "expression" or "gene expression" refers to the process by which information from a gene is used to synthesize a functional gene product. A gene product may be a protein, peptide, or nucleic acid polymer (such as RNA, DNA, or PNA).

如本文所用，“表达盒”是指包含功能性hGLA的编码序列(包括其变体和片段)和启动子的核酸聚合物。在其它实施例中，表达盒除启动子外还包括一个或多个调节序列。在某些实施例中，表达载体是载体基因组。在某些实施例中，表达盒或载体基因组被包装到载体中。在某些实施例中，提供了包括本文所述的表达盒的质粒。As used herein, "expression cassette" refers to a nucleic acid polymer comprising a coding sequence of functional hGLA (including variants and fragments thereof) and a promoter. In other embodiments, the expression cassette includes one or more regulatory sequences in addition to the promoter. In certain embodiments, the expression vector is a vector genome. In certain embodiments, the expression cassette or vector genome is packaged into a vector. In certain embodiments, a plasmid comprising an expression cassette described herein is provided.

如本文所用，术语“调节序列”或“表达控制序列”是指核酸序列，如起始序列、增强子序列和启动子序列，其诱导、抑制或控制与其可操作地连接的编码蛋白质的核酸序列的转录。As used herein, the term "regulatory sequence" or "expression control sequence" refers to nucleic acid sequences, such as initiation sequences, enhancer sequences and promoter sequences, which induce, repress or control the transcription of a protein-encoding nucleic acid sequence to which it is operably linked.

如本文所用，术语“可操作地连接”是指与编码hGLA的核酸序列相邻的表达控制序列和/或以反式或以一定距离起作用以控制其转录和表达的表达控制序列。As used herein, the term "operably linked" refers to an expression control sequence that is adjacent to a nucleic acid sequence encoding hGLA and/or that functions in trans or at a distance to control its transcription and expression.

术语“异源”当用于指质粒、表达盒或载体中的蛋白质或核酸时，表示该蛋白质或核酸与另一序列或亚序列一起存在，该蛋白质或核酸与该另一序列或亚序列在自然界中彼此不存在相同的关系。The term "heterologous" when used in reference to a protein or nucleic acid in a plasmid, expression cassette or vector means that the protein or nucleic acid is present with another sequence or subsequence that is not found in the same relationship to each other in nature.

在某些实施例中，所提供的表达盒包括为鸡β-肌动蛋白启动子的启动子。已经单独或与各种增强子元件(例如，CB7是具有巨细胞病毒增强子元件的鸡β-肌动蛋白启动子、CAG启动子，其包括启动子、鸡β肌动蛋白的第一外显子和第一内含子，以及兔β-珠蛋白基因的剪接受体)、CBh启动子组合描述了多种鸡β-肌动蛋白启动子[SJ Gray等人，《人类基因治疗(Hu Gene Ther)》，2011年9月；22(9)：1143-1153]。在其它实施例中，合适的启动子可以包括但不限于延长因子1α(EF1α)启动子(参见，例如，Kim DW等人，《使用人类延长因子1α启动子作为通用和有效的表达系统(Use of the human elongation factor 1 alphapromoter as a versatile and efficient expression system)》《基因(Gene)》1990年7月16日；91(2)：217-23)、突触蛋白1启动子(参见，例如，Kügler S等人，《人突触蛋白1基因启动子在成年大鼠脑中根据转导区域赋予腺病毒载体高度神经元特异性的长期转基因表达(Human synapsin 1 gene promoter confers highly neuron-specific long-termtransgene expression from an adenoviral vector in the adult rat braindepending on the transduced area)》《基因疗法(Gene Ther.)》2003年2月；10(4)：337-47)、神经元特异性烯醇化酶(NSE)启动子(参见，例如，Kim J等人，《富含胆固醇的脂筏参与白细胞介素-6诱导的LNCaP前列腺癌细胞神经内分泌分化(Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation ofLNCaP prostate cancer cells)》《内分泌学(Endocrinology)》2004年2月；145(2)：613-9.电子版2003年10月16日)，或CB6启动子(参见，例如，《携带人存活运动神经元基因的血清9型腺相关病毒载体的大规模生产(Large-Scale Production of Adeno-Associated ViralVector Serotype-9Carrying the Human Survival Motor Neuron Gene)》，《分子生物技术(Mol Biotechnol.)》2016年1月；58(1)：30-6.doi：10.1007/s12033-015-9899-5)。In certain embodiments, the expression cassettes provided include a promoter that is a chicken β-actin promoter. A variety of chicken β-actin promoters have been described alone or in combination with various enhancer elements (e.g., CB7 is a chicken β-actin promoter with a cytomegalovirus enhancer element, a CAG promoter that includes a promoter, the first exon and the first intron of chicken β-actin, and a splice acceptor of a rabbit β-globin gene), a CBh promoter [SJ Gray et al., Hu Gene Ther, 2011 Sep; 22(9): 1143-1153]. In other embodiments, suitable promoters may include, but are not limited to, the elongation factor 1 alpha (EF1α) promoter (see, e.g., Kim DW et al., Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system, Gene. 1990 Jul 16;91(2):217-23), the synapsin 1 promoter (see, e.g., Kügler S et al., Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area, Gene Ther. 2003 Feb;10(4):337-47), the neuron-specific enolase (NSE ... J et al. Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology. 2004 Feb;145(2):613-9. Epub 2003 Oct 16), or the CB6 promoter (see, e.g., Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene. Mol Biotechnol. 2016 Jan;58(1):30-6. doi: 10.1007/s12033-015-9899-5).

组织特异性启动子的实例对于肝脏和其它组织是熟知的(白蛋白，Miyatakc等人，(1997)《病毒学杂志(J.Virol.)》，71：5124-32；乙型肝炎病毒核心启动子，Sandig等人，(1996)《基因疗法》，3：1002-9；甲胎蛋白(AFP)，Arbuthnot等人，(1996)《人类基因治疗》7：1503-14)，骨骨钙蛋白(Stein等人，(1997)《分子生物学报告(Mol.Biol.Rep.)》，24：185-96)；骨唾液蛋白(Chen等人，(1996)《骨矿物质研究杂志(J.Bone Miner.Res.)》，11：654-64)、淋巴细胞(CD2，Hansal等人，(1998)《免疫学杂志(J.Immunol.)》，161：1063-8；免疫球蛋白重链；T细胞受体链)、神经元(如神经元特异性烯醇化酶(NSE))启动子(Andersen等人，(1993)《细胞分子神经生物杂志(Cell.Mol.Neurobiol.)》，13：503-15)、神经丝轻链基因(Piccioli等人，(1991)《美国国家科学院院刊(Proc.Natl.Acad.Sci.USA)》，88：5611-5)和神经元特异性vgf基因(Piccioli等人，(1995)《神经元(Neuron)》，15：373-84)等。在某些实施例中，启动子是人甲状腺素结合球蛋白(TBG)启动子。替代地，可以选择可调节的启动子。参见，例如，WO 2011/126808B2，其通过引用并入本文。Examples of tissue-specific promoters are well known for liver and other tissues (albumin, Miyatak et al., (1997) J. Virol., 71:5124-32; hepatitis B virus core promoter, Sandig et al., (1996) Gene Therapy, 3:1002-9; alpha-fetoprotein (AFP), Arbuthnot et al., (1996) Human Gene Therapy, 7:1503-14), bone osteocalcin (Stein et al., (1997) Mol. Biol. Rep., 24:185-96); bone sialoprotein (Chen et al., (1996) J. Bone Mineral Res., 24:185-96); Miner. Res., 11: 654-64), lymphocytes (CD2, Hansal et al., (1998) Journal of Immunology (J. Immunol.), 161: 1063-8; immunoglobulin heavy chain; T cell receptor chain), neurons (such as neuron-specific enolase (NSE)) promoter (Andersen et al., (1993) Cell. Mol. Neurobiol., 13: 503-15), neurofilament light chain gene (Piccioli et al., (1991) Proc. Natl. Acad. Sci. USA, 88: 5611-5) and neuron-specific vgf gene (Piccioli et al., (1995) Neuron, 15: 373-84), etc. In certain embodiments, the promoter is human thyroxine binding globulin (TBG) promoter. Alternatively, a regulatable promoter may be selected. See, e.g., WO 2011/126808 B2, which is incorporated herein by reference.

在某些实施例中，表达盒包括一种或多种表达增强子。在某些实施例中，表达盒含有两个或更多个表达增强子。这些增强子可以是相同或不同的。例如，增强子可以包括αmic/bik增强子或CMV增强子。这种增强子可以以彼此相邻的两个拷贝存在。替代地，增强子的双拷贝可以被一个或多个序列分开。在更进一步的实施例中，表达盒进一步含有内含子，例如鸡β-肌动蛋白内含子、人β-球蛋白内含子、SV40内含子和/或商业上可获得的内含子。其它合适的内含子包括本领域已知的那些，例如，如WO 2011/126808中所述的那些。In certain embodiments, the expression cassette includes one or more expression enhancers. In certain embodiments, the expression cassette contains two or more expression enhancers. These enhancers can be the same or different. For example, the enhancer can include an αmic/bik enhancer or a CMV enhancer. Such an enhancer can exist in two copies adjacent to each other. Alternatively, the two copies of the enhancer can be separated by one or more sequences. In further embodiments, the expression cassette further contains an intron, such as a chicken β-actin intron, a human β-globin intron, an SV40 intron, and/or a commercially available Introns. Other suitable introns include those known in the art, for example, as described in WO 2011/126808.

所提供的表达盒可以包括一种或多种表达增强子，如来自土拨鼠肝炎病毒(WPRE)、人肝炎病毒(HPRE)、地松鼠肝炎病毒(GPRE)或北极地松鼠肝炎病毒(AGSPRE)的转录后调节元件；或合成的转录后调节元件。这些表达增强元件当置于3′UTR中时是特别有利的并且可以显著增加mRNA稳定性和/或蛋白质产量。在某些实施例中，所提供的表达盒包括调节序列，其为土拨鼠肝炎病毒转录后调节元件(WPRE)或其变体。合适的WPRE序列提供在本文所述的载体基因组中，并且是本领域已知的(例如，如美国专利第6,136,597号、第6,287,814号和第7,419,829号中所述的那些，这些专利通过引用并入本文)。在某些实施例中，WPRE是一种已经被突变以消除土拨鼠乙型肝炎病毒X(WHX)蛋白的表达的变体，包括例如WHX基因的起始密码子中的突变(参见，Zanta-Boussif等人，《基因疗法》2009年5月；16(5)：605-19，其通过引用并入本文)。在某些实施例中，WPRE包含SEQ ID NO：27中提供的核苷酸序列。在其它实施例中，增强子选自非病毒来源。The provided expression cassettes may include one or more expression enhancers, such as post-transcriptional regulatory elements from woodchuck hepatitis virus (WPRE), human hepatitis virus (HPRE), ground squirrel hepatitis virus (GPRE), or Arctic ground squirrel hepatitis virus (AGSPRE); or synthetic post-transcriptional regulatory elements. These expression enhancing elements are particularly advantageous when placed in the 3′UTR and can significantly increase mRNA stability and/or protein production. In certain embodiments, the provided expression cassettes include a regulatory sequence that is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or a variant thereof. Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., those described in U.S. Pat. Nos. 6,136,597, 6,287,814, and 7,419,829, which are incorporated herein by reference). In certain embodiments, the WPRE is a variant that has been mutated to eliminate expression of the woodchuck hepatitis B virus X (WHX) protein, including, for example, a mutation in the start codon of the WHX gene (see, Zanta-Boussif et al., Gene Therapy 2009 May;16(5):605-19, which is incorporated herein by reference). In certain embodiments, the WPRE comprises the nucleotide sequence provided in SEQ ID NO: 27. In other embodiments, the enhancer is selected from a non-viral source.

此外，所提供的表达盒包括合适的聚腺苷酸化信号。在某些实施例中，polyA序列是兔β-珠蛋白polyA。参见，例如WO 2014/151341。在另一实施例中，polyA序列是牛生长激素polyA。替代地，包括另一种polyA，例如人生长激素(hGH)聚腺苷酸化序列、S450 polyA或合成的polyA。In addition, the provided expression cassettes include a suitable polyadenylation signal. In certain embodiments, the polyA sequence is rabbit β-globin polyA. See, e.g., WO 2014/151341. In another embodiment, the polyA sequence is bovine growth hormone polyA. Alternatively, another polyA is included, such as a human growth hormone (hGH) polyadenylation sequence, S450 polyA, or a synthetic polyA.

在某些实施例中，表达盒可以在非翻译区中包括一个或多个miRNA(也称为miR或微RNA)靶序列。设计miRNA靶序列以被存在于其中转基因表达是不期望的和/或转基因表达水平降低是期望的细胞中的miRNA特异性识别。在某些实施例中，表达盒包括特异性减少背根神经节中hGLA表达的miRNA靶序列。在某些实施例中，miRNA靶序列位于表达盒的3′UTR、5′UTR和/或3′和5′UTR两者中。在某些实施例中，表达盒包含背根神经节(DRG)特异性miRNA靶序列的至少两个串联重复序列，其中至少两个串联重复序列包含可以相同或不同的至少第一miRNA靶序列和至少第二miRNA靶序列。在某些实施例中，至少两个DRG特异性miRNA串联重复序列中的第一个的起点在距hGLA编码序列的3′端20个核苷酸内。在某些实施例中，至少两个DRG特异性miRNA串联重复序列中的第一个的起点距离hGLA编码序列的3′端至少100个核苷酸。在某些实施例中，miRNA串联重复序列包含200至1200个核苷酸的长度。在某些实施例中，相对于缺少miR靶序列的表达盒，包括miR靶不会改变治疗性转基因在一个或多个靶组织中的表达或疗效。In certain embodiments, the expression cassette may include one or more miRNA (also referred to as miR or microRNA) target sequences in the untranslated region. The miRNA target sequence is designed to be specifically recognized by miRNAs present in cells in which transgenic expression is undesirable and/or a reduction in transgenic expression levels is desired. In certain embodiments, the expression cassette includes a miRNA target sequence that specifically reduces hGLA expression in the dorsal root ganglion. In certain embodiments, the miRNA target sequence is located in the 3′UTR, 5′UTR, and/or both 3′ and 5′UTR of the expression cassette. In certain embodiments, the expression cassette comprises at least two tandem repeats of a dorsal root ganglion (DRG)-specific miRNA target sequence, wherein at least two tandem repeats comprise at least a first miRNA target sequence and at least a second miRNA target sequence that may be the same or different. In certain embodiments, the starting point of the first of at least two DRG-specific miRNA tandem repeats is within 20 nucleotides of the 3′ end of the hGLA coding sequence. In certain embodiments, the starting point of the first of at least two DRG-specific miRNA tandem repeats is at least 100 nucleotides from the 3′ end of the hGLA coding sequence. In certain embodiments, the miRNA tandem repeat sequence comprises a length of 200 to 1200 nucleotides. In certain embodiments, including a miR target does not alter expression or efficacy of the therapeutic transgene in one or more target tissues relative to an expression cassette lacking the miR target sequence.

在某些实施例中，表达盒含有至少一种miRNA靶序列，其为miR-183靶序列。在某些实施例中，表达盒含有miR-183靶序列，其包括AGTGAATTCTACCAGTGCCATA(SEQ ID NO：31)，其中与miR-183种子序列互补的序列用下划线标出。在某些实施例中，表达盒含有多于一个拷贝(例如两个或三个拷贝)的与miR-183种子序列100％互补的序列。在某些实施例中，miR-183靶序列长度为约7个核苷酸至约28个核苷酸并且包括至少一个与miR-183种子序列至少100％互补的区域。在某些实施例中，miR-183靶序列含有与SEQ ID NO：31具有部分互补性的序列，因此，当与SEQ ID NO：31比对时，存在一个或多个错配。在某些实施例中，miR-183靶序列包含当与SEQ ID NO：31比对时具有至少1、2、3、4、5、6、7、8、9或10个错配的序列，其中错配可以是非邻接的。在某些实施例中，miR-183靶序列包括100％互补性的区域，其也包含miR-183靶序列长度的至少30％。在某些实施例中，100％互补性的区域包括与miR-183种子序列具有100％互补性的序列。在某些实施例中，miR-183靶序列的剩余部分与miR-183具有至少约80％至约99％的互补性。在某些实施例中，表达盒包括miR-183靶序列，其包含截短的SEQ ID NO：31，即，在SEQ ID NO：31的5′端或3′端中的任一端或两端缺失至少1、2、3、4、5、6、7、8、9或10个核苷酸的序列。在某些实施例中，表达盒包含转基因和一个miR-183靶序列。在其它实施例中，表达盒包含至少两个、三个或四个miR-183靶序列。在某些实施例中，在表达盒中包括至少两个、三个或四个miR-183靶序列导致靶组织(如心脏)中转基因表达水平增加。In certain embodiments, the expression cassette contains at least one miRNA target sequence that is a miR-183 target sequence. In certain embodiments, the expression cassette contains a miR-183 target sequence that includes AGTGAATTCTCACCAGTGCCAT A (SEQ ID NO: 31), wherein the sequence complementary to the miR-183 seed sequence is underlined. In certain embodiments, the expression cassette contains more than one copy (e.g., two or three copies) of a sequence that is 100% complementary to the miR-183 seed sequence. In certain embodiments, the miR-183 target sequence is about 7 nucleotides to about 28 nucleotides in length and includes at least one region that is at least 100% complementary to the miR-183 seed sequence. In certain embodiments, the miR-183 target sequence contains a sequence that is partially complementary to SEQ ID NO: 31, and therefore, when aligned with SEQ ID NO: 31, there are one or more mismatches. In certain embodiments, the miR-183 target sequence comprises a sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches when aligned with SEQ ID NO: 31, wherein the mismatches may be non-contiguous. In certain embodiments, the miR-183 target sequence comprises a region of 100% complementarity that also comprises at least 30% of the length of the miR-183 target sequence. In certain embodiments, the region of 100% complementarity comprises a sequence having 100% complementarity with a miR-183 seed sequence. In certain embodiments, the remainder of the miR-183 target sequence has at least about 80% to about 99% complementarity with miR-183. In certain embodiments, the expression cassette comprises a miR-183 target sequence comprising a truncated SEQ ID NO: 31, i.e., a sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides missing from either or both of the 5′ or 3′ ends of SEQ ID NO: 31. In certain embodiments, the expression cassette comprises a transgene and a miR-183 target sequence. In other embodiments, the expression cassette comprises at least two, three or four miR-183 target sequences. In certain embodiments, including at least two, three or four miR-183 target sequences in the expression cassette results in an increase in the level of transgene expression in a target tissue such as the heart.

在某些实施例中，表达盒含有至少一种miRNA靶序列，其为miR-182靶序列。在某些实施例中，表达盒含有miR-182靶序列，其包括AGTGTGAGTTCTACCATTGCCAAA(SEQ ID NO：32)。在某些实施例中，表达盒含有多于一个拷贝(例如两个或三个拷贝)的与miR-182种了序列100％互补的序列。在某些实施例中，miR-182靶序列长度为约7个核苷酸至约28个核苷酸并且包括至少一个与miR-182种子序列至少100％互补的区域。在某些实施例中，miR-182靶序列含有与SEQ ID NO：32具有部分互补性的序列，因此，当与SEQ ID NO：32比对时，存在一个或多个错配。在某些实施例中，miR-183靶序列包含当与SEQ ID NO：32比对时具有至少1、2、3、4、5、6、7、8、9或10个错配的序列，其中错配可以是非邻接的。在某些实施例中，miR-182靶序列包括100％互补性的区域，其也包含miR-182靶序列长度的至少30％。在某些实施例中，100％互补性的区域包括与miR-182种子序列具有100％互补性的序列。在某些实施例中，miR-182靶序列的剩余部分与miR-182具有至少约80％至约99％的互补性。在某些实施例中，表达盒包括miR-182靶序列，其包含截短的SEQ ID NO：32，即，在SEQ ID NO：32的5′端或3′端中的任一端或两端缺失至少1、2、3、4、5、6、7、8、9或10个核苷酸的序列。在某些实施例中，表达盒包含转基因和一个miR-182靶序列。在其它实施例中，表达盒包含至少两个、三个或四个miR-182靶序列。In certain embodiments, the expression cassette contains at least one miRNA target sequence that is a miR-182 target sequence. In certain embodiments, the expression cassette contains a miR-182 target sequence that includes AGTGTGAGTTTCTACCATTGCCAAA (SEQ ID NO: 32). In certain embodiments, the expression cassette contains more than one copy (e.g., two or three copies) of a sequence that is 100% complementary to the miR-182 seed sequence. In certain embodiments, the miR-182 target sequence is about 7 nucleotides to about 28 nucleotides in length and includes at least one region that is at least 100% complementary to the miR-182 seed sequence. In certain embodiments, the miR-182 target sequence contains a sequence that is partially complementary to SEQ ID NO: 32, and therefore, when aligned with SEQ ID NO: 32, there are one or more mismatches. In certain embodiments, the miR-183 target sequence comprises a sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches when aligned with SEQ ID NO: 32, wherein the mismatches may be non-contiguous. In certain embodiments, the miR-182 target sequence comprises a region of 100% complementarity that also comprises at least 30% of the length of the miR-182 target sequence. In certain embodiments, the region of 100% complementarity comprises a sequence having 100% complementarity with a miR-182 seed sequence. In certain embodiments, the remainder of the miR-182 target sequence has at least about 80% to about 99% complementarity with miR-182. In certain embodiments, the expression cassette comprises a miR-182 target sequence comprising a truncated SEQ ID NO: 32, i.e., a sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides missing from either or both of the 5′ or 3′ ends of SEQ ID NO: 32. In certain embodiments, the expression cassette comprises a transgene and one miR-182 target sequence. In other embodiments, the expression cassette comprises at least two, three or four miR-182 target sequences.

术语“串联重复序列”在本文中用于指两个或更多个连续miRNA靶序列的存在。这些miRNA靶序列可以是连续的，即，直接位于彼此之后，使得一个的3′端直接位于下一个的5′端的上游，没有插入序列，反之亦然。在另一实施例中，两个或更多个miRNA靶序列被短间隔区序列分开。The term "tandem repeat sequence" is used herein to refer to the presence of two or more consecutive miRNA target sequences. These miRNA target sequences can be consecutive, that is, directly after each other, so that the 3' end of one is directly upstream of the 5' end of the next, without intervening sequences, or vice versa. In another embodiment, the two or more miRNA target sequences are separated by a short spacer sequence.

如本文所用，“间隔区”是位于两个或更多个连续miRNA靶序列之间的任何选定的核酸序列，例如长度为1、2、3、4、5、6、7、8、9或10个核苷酸。在某些实施例中，间隔区长度为1至8个核苷酸、长度为2至7个核苷酸、长度为3至6个核苷酸、长度为四个核苷酸、4至9个核苷酸、3至7个核苷酸或更长的值。合适地，间隔区是非编码序列。在某些实施例中，间隔区可以是四(4)个核苷酸。在某些实施例中，间隔区是GGAT。在某些实施例中，间隔区是六(6)个核苷酸。在某些实施例中，间隔区是CACGTG或GCATGC。As used herein, a "spacer" is any selected nucleic acid sequence located between two or more consecutive miRNA target sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In certain embodiments, the spacer is 1 to 8 nucleotides in length, 2 to 7 nucleotides in length, 3 to 6 nucleotides in length, four nucleotides in length, 4 to 9 nucleotides, 3 to 7 nucleotides or longer. Suitably, the spacer is a non-coding sequence. In certain embodiments, the spacer can be four (4) nucleotides. In certain embodiments, the spacer is GGAT. In certain embodiments, the spacer is six (6) nucleotides. In certain embodiments, the spacer is CACGTG or GCATGC.

在某些实施例中，串联重复序列含有两个、三个、四个或更多个相同的miRNA靶序列。在某些实施例中，串联重复序列含有至少两种不同的miRNA靶序列、至少三种不同的miRNA靶序列或至少四种不同的miRNA靶序列等。在某些实施例中，串联重复序列可以含有两个或三个相同的miRNA靶序列和不同的第四个miRNA靶序列。In certain embodiments, the tandem repeat sequence contains two, three, four or more identical miRNA target sequences. In certain embodiments, the tandem repeat sequence contains at least two different miRNA target sequences, at least three different miRNA target sequences, or at least four different miRNA target sequences, etc. In certain embodiments, the tandem repeat sequence may contain two or three identical miRNA target sequences and a different fourth miRNA target sequence.

在某些实施例中，在表达盒中可以存在至少两组不同的串联重复序列。例如，3′UTR可以含有紧接转基因下游的串联重复序列、UTR序列，以及两个或更多个靠近UTR的3′端的串联重复序列。在另一实例中，5′UTR可以含有一个、两个或更多个miRNA靶序列。在另一实例中，3′UTR可以含有串联重复序列且5′UTR可以含有至少一个miRNA靶序列。In certain embodiments, at least two different sets of tandem repeat sequences may be present in the expression cassette. For example, the 3'UTR may contain a tandem repeat sequence immediately downstream of the transgene, a UTR sequence, and two or more tandem repeat sequences near the 3' end of the UTR. In another example, the 5'UTR may contain one, two or more miRNA target sequences. In another example, the 3'UTR may contain a tandem repeat sequence and the 5'UTR may contain at least one miRNA target sequence.

在某些实施例中，表达盒含有两个、三个、四个或更多个串联重复序列，其起始于转基因的终止密码子的约0至20个核苷酸内。在其它实施例中，表达盒含有距离转基因终止密码子至少100至约4000个核菅酸的miRNA串联重复序列。In certain embodiments, the expression cassette contains two, three, four or more tandem repeats starting within about 0 to 20 nucleotides of the stop codon of the transgene. In other embodiments, the expression cassette contains a miRNA tandem repeat at least 100 to about 4000 nucleotides from the stop codon of the transgene.

还参见2019年12月20日提交的国际专利申请号PCT/US19/67872和2021年5月12日提交的国际专利申请号PCT/US21/32003，其全部内容通过引用并入本文。See also International Patent Application No. PCT/US19/67872 filed on December 20, 2019 and International Patent Application No. PCT/US21/32003 filed on May 12, 2021, the entire contents of which are incorporated herein by reference.

应当理解，表达盒中的组合物旨在应用于说明书中描述的其它组合物、方案、方面、实施例和方法。It should be understood that the compositions in the expression cassettes are intended to be applied to other compositions, schemes, aspects, embodiments and methods described in the specification.

3.载体3. Carrier

在一个方面，本文提供了包含编码功能性hGLA的核酸序列的载体。在某些实施例中，载体包含如本文所述的用于递送hGLA编码序列的表达盒。In one aspect, provided herein are vectors comprising a nucleic acid sequence encoding functional hGLA. In certain embodiments, the vector comprises an expression cassette as described herein for delivering an hGLA coding sequence.

本文所用的“载体”是包含核酸序列的生物或化学部分，这些核酸序列可以被引入合适的靶细胞中用于所述核酸序列的复制或表达。载体的实例包括但不限于重组病毒、质粒、脂质体、多聚体、多聚物、树状聚体、细胞穿透肽(CPP)缀合物、磁性颗粒或纳米颗粒。在某些实施例中，载体是其中可以插入编码功能性hGLA的工程化核酸的核酸分子，然后可以将其引入合适的靶细胞中。这些载体优选地具有一个或多个复制起点，以及一个或多个重组DNA可插入的位点。载体通常具有一些手段，通过这些手段可以将具有载体的细胞从没有载体的细胞中选择出来，例如，它们编码抗药性基因。常见的载体包括质粒、病毒基因组和“人工染色体”。本领域技术人员可以获得生成、生产、表征或定量载体的常规方法As used herein, "vector" is a biological or chemical part containing a nucleic acid sequence that can be introduced into a suitable target cell for replication or expression of the nucleic acid sequence. Examples of vectors include, but are not limited to, recombinant viruses, plasmids, liposomes, polymers, polymers, dendrimers, cell penetrating peptide (CPP) conjugates, magnetic particles or nanoparticles. In certain embodiments, a vector is a nucleic acid molecule into which an engineered nucleic acid encoding functional hGLA can be inserted, which can then be introduced into a suitable target cell. These vectors preferably have one or more origins of replication, and one or more sites into which recombinant DNA can be inserted. Vectors typically have some means by which cells with the vector can be selected from cells without the vector, for example, they encode drug resistance genes. Common vectors include plasmids, viral genomes and "artificial chromosomes". Conventional methods for generating, producing, characterizing or quantifying vectors are available to those skilled in the art.

在某些实施例中，载体是非病毒质粒，其包含本文所述的表达盒(例如，“裸DNA”、“裸质粒DNA”、RNA和mRNA，其可以与各种组合物和纳米颗粒偶联，包括例如胶束、脂质体、阳离子脂质-核酸组合物、聚糖组合物和其它聚合物、基于脂质和/或胆固醇的核酸缀合物)和如本文所述的其它构建体。参见，例如，X.Su等人，《分子药剂学(Mol.Pharmaceutics)》，2011，8(3)，第774至787页；网络出版：2011年3月21日；WO2013/182683、WO 2010/053572和WO 2012/170930，所有这些通过引用并入本文。In certain embodiments, the vector is a non-viral plasmid comprising an expression cassette described herein (e.g., "naked DNA," "naked plasmid DNA," RNA and mRNA, which can be coupled to various compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, polysaccharide compositions and other polymers, lipid- and/or cholesterol-based nucleic acid conjugates) and other constructs as described herein. See, e.g., X. Su et al., Mol. Pharmaceutics, 2011, 8(3), pp. 774-787; Online publication: Mar. 21, 2011; WO 2013/182683, WO 2010/053572, and WO 2012/170930, all of which are incorporated herein by reference.

在某些实施例中，本文所述的载体是“复制缺陷型病毒”或“病毒载体”，其是指合成或人工病毒颗粒，其中含有编码hGLA的核酸序列的表达盒包装在病毒衣壳或包膜中，其中也包装在病毒衣壳或包膜内的任何病毒基因组序列是复制缺陷型的；即，它们不能生成子代病毒体，但保留感染靶细胞的能力。在一个实施例中，病毒载体的基因组不包括编码复制所需的酶的基因(基因组可以被工程化为“无细胞的”——仅含有编码hGLA的核酸序列，其侧翼为扩增和包装人工基因组所需的信号)，但这些基因可以在生产期间提供。因此，它被认为可以安全地用于基因治疗，因为子代病毒体的复制和感染只有在复制所需的病毒酶存在时才会发生。In certain embodiments, the vectors described herein are "replication-defective viruses" or "viral vectors," which refer to synthetic or artificial viral particles in which an expression cassette containing a nucleic acid sequence encoding hGLA is packaged in a viral capsid or envelope, wherein any viral genomic sequences also packaged within the viral capsid or envelope are replication-defective; that is, they are unable to generate progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding enzymes required for replication (the genome can be engineered to be "cell-free" - containing only the nucleic acid sequence encoding hGLA, flanked by signals required for amplification and packaging of the artificial genome), but these genes can be provided during production. Therefore, it is considered safe for use in gene therapy because replication and infection of progeny virions will only occur when the viral enzymes required for replication are present.

如本文所用，重组病毒载体是腺相关病毒(AAV)、腺病毒、博卡病毒、杂合AAV/博卡病毒、单纯疱疹病毒或慢病毒。As used herein, a recombinant viral vector is an adeno-associated virus (AAV), an adenovirus, a bocavirus, a hybrid AAV/bocavirus, a herpes simplex virus, or a lentivirus.

在某些实施例中，提供了具有包括hGLA编码序列的核酸的宿主细胞。在某些实施例中，宿主细胞含有具有如本文所述的hGLA编码序列的质粒。In certain embodiments, a host cell having a nucleic acid comprising an hGLA coding sequence is provided. In certain embodiments, a host cell contains a plasmid having an hGLA coding sequence as described herein.

如本文所用，术语“宿主细胞”可以指其中产生载体(例如，重组AAV)的包装细胞系。宿主细胞可以是含有外源或异源DNA的原核或真核细胞(例如，人、昆虫或酵母)，外源或异源DNA已通过任何方式引入细胞中，例如电穿孔、磷酸钙沉淀、显微注射、转化、病毒感染、转染、脂质体递送、膜融合技术、高速DNA包被颗粒、病毒感染和原生质体融合。宿主细胞的实例可以包括但不限于分离的细胞、细胞培养物、大肠杆菌(Escherichia coli)细胞、酵母细胞、人细胞、非人细胞、哺乳动物细胞、非哺乳动物细胞、昆虫细胞、HEK-293细胞、肝细胞、肾细胞、中枢神经系统细胞、神经元、神经胶质细胞或干细胞。As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., recombinant AAV) is produced. A host cell may be a prokaryotic or eukaryotic cell (e.g., human, insect, or yeast) containing exogenous or heterologous DNA, which has been introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion technology, high-speed DNA coated particles, viral infection, and protoplast fusion. Examples of host cells may include, but are not limited to, isolated cells, cell cultures, Escherichia coli cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK-293 cells, hepatocytes, kidney cells, central nervous system cells, neurons, glial cells, or stem cells.

在某些实施例中，宿主细胞含有用于产生hGLA的表达盒，使得在体外以足够的量产生蛋白质用于分离或纯化。在某些实施例中，宿主细胞含有编码hGLA(包括例如其功能片段)的表达盒。如本文提供的，hGLA多肽可以包括在作为治疗剂(即，酶替代疗法)施用于受试者的药物组合物中。In certain embodiments, the host cell contains an expression cassette for producing hGLA, so that the protein is produced in sufficient quantity in vitro for isolation or purification. In certain embodiments, the host cell contains an expression cassette encoding hGLA (including, for example, its functional fragments). As provided herein, the hGLA polypeptide can be included in a pharmaceutical composition administered to a subject as a therapeutic agent (i.e., enzyme replacement therapy).

如本文所用，术语“靶细胞”是指其中期望表达功能性hGLA的任何细胞。在某些实施例中，术语“靶细胞”旨在指正在接受法布里病治疗的受试者的细胞。靶细胞的实例可以包括但不限于肝细胞、肾细胞、平滑肌细胞和神经元。在某些实施例中，将载体离体递送至靶细胞。在某些实施例中，在体内将载体递送至靶细胞。As used herein, the term "target cell" refers to any cell in which expression of functional hGLA is desired. In certain embodiments, the term "target cell" is intended to refer to a cell of a subject being treated for Fabry disease. Examples of target cells may include, but are not limited to, hepatocytes, kidney cells, smooth muscle cells, and neurons. In certain embodiments, the vector is delivered to the target cell ex vivo. In certain embodiments, the vector is delivered to the target cell in vivo.

应当理解，本文所述载体中的组合物旨在应用于说明书中描述的其它组合物、方案、方面、实施例和方法。It is to be understood that the compositions in the vectors described herein are intended to be applicable to other compositions, schemes, aspects, embodiments and methods described in the specification.

4.重组腺相关病毒(rAAV)4. Recombinant adeno-associated virus (rAAV)

在某些实施例中，本文提供了包含AAV衣壳和包装在其中的载体基因组的rAAV。载体基因组包含AAV 5′反向末端重复序列(ITR)、编码本文所述的功能性hGLA的核酸序列、指导hGLA在靶细胞中表达的调节序列和AAV 3′ITR。在某些实施例中，载体基因组包含如本文提供的表达盒，其侧翼为AAV 5′ITR和AAV 3′ITR。这种rAAV适用于治疗法布里病。In certain embodiments, provided herein is a rAAV comprising an AAV capsid and a vector genome packaged therein. The vector genome comprises an AAV 5′ inverted terminal repeat (ITR), a nucleic acid sequence encoding a functional hGLA as described herein, a regulatory sequence for directing hGLA expression in a target cell, and an AAV 3′ITR. In certain embodiments, the vector genome comprises an expression cassette as provided herein, flanked by an AAV 5′ITR and an AAV 3′ITR. This rAAV is suitable for treating Fabry disease.

如本文所用，“rAAV.hGLA”是指具有包括hGLA编码序列的载体基因组的rAAV。“rAAVhu68.hGLA”是指具有AAVhu68衣壳和包括hGLA编码序列的载体基因组的rAAV。As used herein, "rAAV.hGLA" refers to rAAV having a vector genome including the hGLA coding sequence. "rAAVhu68.hGLA" refers to rAAV having an AAVhu68 capsid and a vector genome including the hGLA coding sequence.

如本文所用，“载体基因组”是指包装在载体内的核酸序列。在一个实施例中，载体基因组是指包装在rAAV衣壳内形成rAAV载体的核酸序列。这种核酸序列含有AAV反向末端重复序列(ITR)。在某些实施例中，ITR来自不同于提供衣壳的AAV。在一个优选的实施例中，来自AAV2的ITR序列或其缺失版本(ΔITR)可用于方便和加速监管批准。然而，可以选择来自其它AAV来源的ITR。当ITR的来源来自AAV2而AAV衣壳来自另一AAV来源时，所得载体可以称为假型载体。通常，AAV载体基因组包含AAV 5′ITR、调节序列、hGLA编码序列和AAV 3′ITR。然而，这些元件的其它构造也是合适的。已经描述了5′ITR的缩短版本，称为AITR，其中缺失了D序列和末端解析位点(trs)。在某些实施例中，载体基因组包括130个碱基对的缩短的AAV2 ITR，其中缺失了外部A元件。在使用内部A元件作为模板的载体DNA扩增过程中，缩短的ITR回复到145个碱基对的野生型长度。在其它实施例中，使用全长AAV 5′和3′ITR。在某些实施例中，载体基因组包括一个或多个miRNA靶序列。As used herein, "vector genome" refers to a nucleic acid sequence packaged in a vector. In one embodiment, a vector genome refers to a nucleic acid sequence packaged in an rAAV capsid to form an rAAV vector. This nucleic acid sequence contains an AAV inverted terminal repeat (ITR). In certain embodiments, ITR is from an AAV different from the capsid provided. In a preferred embodiment, an ITR sequence from AAV2 or a deleted version thereof (ΔITR) can be used to facilitate and accelerate regulatory approval. However, ITRs from other AAV sources can be selected. When the source of ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as a pseudotype vector. Typically, the AAV vector genome comprises AAV 5'ITR, a regulatory sequence, an hGLA coding sequence, and AAV 3'ITR. However, other configurations of these elements are also suitable. A shortened version of 5'ITR has been described, referred to as AITR, in which a D sequence and a terminal resolution site (trs) are deleted. In certain embodiments, the vector genome includes a shortened AAV2 ITR of 130 base pairs, in which an external A element is deleted. During amplification of the vector DNA using the internal A element as a template, the shortened ITR reverts to the wild-type length of 145 base pairs. In other embodiments, full-length AAV 5' and 3' ITRs are used. In certain embodiments, the vector genome includes one or more miRNA target sequences.

在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、启动子、hGLA编码序列、poly A序列和AAV 3′ITR。在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、启动子、内含子、hGLA编码序列、poly A序列和AAV3′ITR。在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、启动子、hGLA编码序列、WPRE、poly A序列和AAV 3′ITR。在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、启动子、内含子、hGLA编码序列、WPRE、poly A序列和AAV 3′ITR。在某些实施例中，载体基因组具有米自非病毒来源的增强子代替WPRE元件。In certain embodiments, rAAV is provided having a vector genome comprising AAV 5′ITR, a promoter, an hGLA coding sequence, a poly A sequence, and AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome comprising AAV 5′ITR, a promoter, an intron, an hGLA coding sequence, a poly A sequence, and AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome comprising AAV 5′ITR, a promoter, an hGLA coding sequence, a WPRE, a poly A sequence, and AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome comprising AAV 5′ITR, a promoter, an intron, an hGLA coding sequence, a WPRE, a poly A sequence, and AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome comprising AAV 5′ITR, a promoter, an intron, an hGLA coding sequence, a WPRE, a poly A sequence, and AAV 3′ITR. In certain embodiments, the vector genome has an enhancer from a non-viral source in place of the WPRE element.

在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、鸡β-肌动蛋白内含子、hGLA编码序列、WPRE、poly A序列和AAV 3′ITR。在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、CB7启动子、鸡β-肌动蛋白内含子、hGLA编码序列、WPRE、兔β珠蛋白poly A序列和AAV 3′ITR。在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、TBG启动子、鸡β-肌动蛋白内含子、hGLA编码序列、WPRE、牛生长激素poly A序列和AAV 3′ITR。在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、TBG启动子、SV40内含了、hGLA编码序列、WPRE、牛生长激素poly A序列和AAV3′ITR。在某些实施例中，载体基因组具有来自非病毒来源的增强子代替WPRE元件。In certain embodiments, rAAV is provided having a vector genome comprising AAV 5′ITR, chicken β-actin intron, hGLA coding sequence, WPRE, poly A sequence, and AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome comprising AAV 5′ITR, CB7 promoter, chicken β-actin intron, hGLA coding sequence, WPRE, rabbit β-globin poly A sequence, and AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome comprising AAV 5′ITR, TBG promoter, chicken β-actin intron, hGLA coding sequence, WPRE, bovine growth hormone poly A sequence, and AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome comprising AAV 5′ITR, TBG promoter, SV40 intron, hGLA coding sequence, WPRE, bovine growth hormone poly A sequence, and AAV 3′ITR. In certain embodiments, the vector genome has an enhancer from a non-viral source in place of the WPRE element.

在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、启动子、鸡β-肌动蛋白内含子、hGLA编码序列、poly A序列和AAV 3′ITR。在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、CB7启动子、鸡β-肌动蛋白内含子、hGLA编码序列、兔珠蛋白poly A序列和AAV 3′ITR。在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、TBG启动子、鸡β-肌动蛋白内含子、hGLA编码序列、牛生长激素poly A序列和AAV 3′ITR。在某些实施例中，提供了具有载体基因组的rAAV，该载体基因组包括AAV 5′ITR、TBG启动子、SV40内含子、hGLA编码序列、牛生长激素polyA序列和AAV 3′ITR。In certain embodiments, rAAV is provided having a vector genome, the vector genome comprising AAV 5′ITR, a promoter, a chicken β-actin intron, an hGLA coding sequence, a poly A sequence, and an AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome, the vector genome comprising AAV 5′ITR, a CB7 promoter, a chicken β-actin intron, an hGLA coding sequence, a rabbit globin poly A sequence, and an AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome, the vector genome comprising AAV 5′ITR, a TBG promoter, a chicken β-actin intron, an hGLA coding sequence, a bovine growth hormone poly A sequence, and an AAV 3′ITR. In certain embodiments, rAAV is provided having a vector genome, the vector genome comprising AAV 5′ITR, a TBG promoter, a chicken β-actin intron, an hGLA coding sequence, a bovine growth hormone poly A sequence, and an AAV 3′ITR.

在一个实施例中，提供了rAAV，其具有SEQ ID NO：6、8、10、12、14、16或18中所示的载体基因组，或与其至少85％相同的序列。In one embodiment, a rAAV is provided having a vector genome as set forth in SEQ ID NO: 6, 8, 10, 12, 14, 16 or 18, or a sequence at least 85% identical thereto.

如本文所用，可互换使用的术语“rAAV”和“人工AAV”是指但不限于包含衣壳蛋白和包装在其中的载体基因组的AAV，其中载体基因组包含与AAV异源的核酸。在一个实施例中，衣壳蛋白是非大然存在的衣壳。这种人工衣壳可以通过任何合适的技术生成，使用选择的AAV序列(例如，vp1衣壳蛋白的片段)与异源序列组合，这些异源序列可以从不同的选择的AAV、相同AAV的非连续部分、从非AAV病毒来源，或从非病毒来源获得。人工AAV可以是但不限于假型AAV、嵌合AAV衣壳、重组AAV衣壳或“人源化”AAV衣壳。假型载体在本发明中是有用的，其中一种AAV的衣壳被异源衣壳蛋白替代。在一个实施例中，AAV2/5和AAV2/8是示例性假型载体。所选择的遗传元件可以通过任何合适的方法递送，包括转染、电穿孔、脂质体递送、膜融合技术、高速DNA包被颗粒、病毒感染和原生质体融合。用于制备这种构建体的方法是核酸操作领域技术人员已知的，并且包括基因工程、重组工程和合成技术。参见，例如，Green和Sambrook，《分子克隆：实验室手册》，冷泉港出版社，纽约冷泉港(2012)。As used herein, the interchangeable terms "rAAV" and "artificial AAV" refer to, but are not limited to, AAV comprising a capsid protein and a vector genome packaged therein, wherein the vector genome comprises a nucleic acid heterologous to AAV. In one embodiment, the capsid protein is a capsid that does not exist naturally. This artificial capsid can be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence, which can be obtained from different selected AAVs, non-continuous parts of the same AAV, from non-AAV viral sources, or from non-viral sources. Artificial AAV can be, but is not limited to, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Pseudotype vectors are useful in the present invention, in which the capsid of one AAV is replaced by a heterologous capsid protein. In one embodiment, AAV2/5 and AAV2/8 are exemplary pseudotype vectors. The selected genetic elements can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA coated particles, viral infection, and protoplast fusion. Methods for preparing such constructs are known to those skilled in the art of nucleic acid manipulation, and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (2012).

本文所用的术语“AAV”是指天然存在的腺相关病毒、本领域技术人员可获得的和/或根据本文所述的组合物和方法获得的腺相关病毒，以及人工AAV。腺相关病毒(AAV)病毒载体是具有AAV蛋白衣壳的抗AAV DNase颗粒，其中包装有侧翼为AAV反向末端重复序列(ITR)的表达盒，用于递送至靶细胞。AAV衣壳由60个衣壳(cap)蛋白亚基VP1、VP2和VP3组成，它们以二十面体对称排列，比例约为1∶1∶10至1∶1∶20，这取决于所选择的AAV。可以选择各种AAV作为如上鉴定的AAV病毒载体的衣壳来源。参见，例如，美国公开专利申请号2007-0036760-A1；美国公开专利申请号2009-0197338-A1；EP 1310571。还参见，WO 2003/042397(AAV7和其它猿AAV)、美国专利7790449和美国专利7282199(AAV8)、WO 2005/033321和US7,906,111(AAV9)，和WO 2006/110689，和WO 2003/042397(rh.10)。这些文献还描述了可被选择用于生成AAV的其它AAV，并通过引用并入本文。在从人或非人灵长类动物(NHP)中分离或工程化并充分表征的AAV中，人AAV2是作为基因转移载体开发的第一AAV；它已广泛用于不同靶组织和动物模型中的高效基因转移实验。除非另有说明，本文所述的AAV衣壳、ITR和其它选择的AAV组分可以容易地选自任何AAV，包括但不限于通常鉴定为AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV8bp、AAV7M8和AAVAnc80、AAVhu68的AAV，以及任何已知或提及的AAV或尚未发现的AAV的变体或其变体或混合物。AAV9衣壳包括具有衣壳蛋白的rAAV，该衣壳蛋白包含与AAS9926499％相同的氨基酸序列。另参见，US7906111和WO 2005/033321。具有AVVhu68衣壳的rAAV描述于例如WO 2018/160582中，其通过引用并入本文。在某些实施例中，衣壳蛋白由rAAV载体名称中术语“AAV”后的数字或数字和字母的组合指定。另参见2019年2月27日提交的PCT/US19/19804和PCT/US19/19861，各自题为《新型腺相关病毒(AAV)载体、具有减少的衣壳脱酰胺作用的AAV载体及其用途(NovelAdeno-AssociatedVirus(AAV)Vectors，AAV Vectors Having Reduced Capsid Deamidation And UsesTherefor)》，其通过引用整体并入本文。The term "AAV" as used herein refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art and/or obtained according to the compositions and methods described herein, and artificial AAVs. Adeno-associated virus (AAV) viral vectors are AAV DNase-resistant particles with AAV protein capsids, in which expression cassettes flanked by AAV inverted terminal repeats (ITRs) are packaged for delivery to target cells. The AAV capsid consists of 60 capsid (cap) protein subunits VP1, VP2, and VP3, which are arranged in icosahedral symmetry in a ratio of about 1:1:10 to 1:1:20, depending on the AAV selected. Various AAVs can be selected as the capsid source of the AAV viral vector identified above. See, for example, U.S. Published Patent Application No. 2007-0036760-A1; U.S. Published Patent Application No. 2009-0197338-A1; EP 1310571. See also, WO 2003/042397 (AAV7 and other simian AAVs), U.S. Pat. No. 7,790,449 and U.S. Pat. No. 7,282,199 (AAV8), WO 2005/033321 and US Pat. No. 7,906,111 (AAV9), and WO 2006/110689, and WO 2003/042397 (rh.10). These documents also describe other AAVs that can be selected for the generation of AAVs and are incorporated herein by reference. Among the AAVs isolated or engineered and well characterized from humans or non-human primates (NHPs), human AAV2 was the first AAV developed as a gene transfer vector; it has been widely used in efficient gene transfer experiments in different target tissues and animal models. Unless otherwise indicated, the AAV capsids, ITRs, and other selected AAV components described herein can be easily selected from any AAV, including but not limited to AAVs generally identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and AAVAnc80, AAVhu68, and any known or mentioned AAV or AAV variants or mixtures thereof that have not yet been discovered. The AAV9 capsid includes rAAVs having a capsid protein comprising an amino acid sequence identical to AAS9926499%. See also, US7906111 and WO 2005/033321. rAAVs having AVVhu68 capsids are described in, for example, WO 2018/160582, which is incorporated herein by reference. In certain embodiments, the capsid protein is specified by a number or a combination of numbers and letters after the term "AAV" in the name of the rAAV vector. See also PCT/US19/19804 and PCT/US19/19861, filed on February 27, 2019, each entitled “Novel Adeno-Associated Virus (AAV) Vectors, AAV Vectors Having Reduced Capsid Deamidation And Uses Therefor,” which are incorporated herein by reference in their entirety.

如本文所用，关于AAV，术语“变体”是指衍生自已知AAV序列的任何AAV序列，包括具有保守氨基酸置换的那些，和与氨基酸或核酸序列共有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、至少99％或更大序列同一性的那些。在另一实施例中，AAV衣壳包括变体，其可以包括来自任何所述或已知AAV衣壳序列的至多约10％的变异。即，AAV衣壳与本文提供的和/或本领域已知的AAV衣壳共有约90％同一性至约99.9％同一性、约95％至约99％同一性或约97％至约98％同一性。在一个实施例中，AAV衣壳与AAV衣壳共有至少95％的同一性。当测定AAV衣壳的同一性百分比时，可以对任何可变蛋白(例如，vp1、vp2或vp3)进行比较。本文所用的“AAV9变体”包括在例如WO2016/049230、US 8,927,514、US 2015/0344911和US 8,734,809中描述的那些。As used herein, with respect to AAV, the term "variant" refers to any AAV sequence derived from a known AAV sequence, including those with conservative amino acid substitutions, and those having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity with an amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes a variant, which may include up to about 10% variation from any of the described or known AAV capsid sequences. That is, the AAV capsid has about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with the AAV capsid provided herein and/or known in the art. In one embodiment, the AAV capsid has at least 95% identity with the AAV capsid. When determining the percent identity of the AAV capsid, any variable protein (e.g., vp1, vp2, or vp3) can be compared. "AAV9 variants" as used herein include those described in, for example, WO2016/049230, US 8,927,514, US 2015/0344911, and US 8,734,809.

在某些实施例中，AAV衣壳选自天然和工程化的进化枝F腺相关病毒。在某些实施例中，本文提供的rAAV包含AAVhu68衣壳。AAVhu68在进化枝F.AAVhu68(SEQ ID NO：21)之内，与另一进化枝F病毒AAV9不同之处在于vp1的位置67和位置157处的两个编码氨基酸。相反，其它进化枝FAAV(AAV9、hu31、hu32)在位置67处具有Ala，在位置157处具有Ala。然而，在其它实施例中，AAV衣壳选自不同的进化枝，例如进化枝A、B、C、D或E，或选自任何这些进化枝之外的AAV来源。In certain embodiments, the AAV capsid is selected from natural and engineered clade F adeno-associated viruses. In certain embodiments, the rAAV provided herein comprises an AAVhu68 capsid. AAVhu68 is within clade F. AAVhu68 (SEQ ID NO: 21) differs from another clade F virus, AAV9, by two encoded amino acids at position 67 and position 157 of vp1. In contrast, other clade FAAVs (AAV9, hu31, hu32) have Ala at position 67 and Ala at position 157. However, in other embodiments, the AAV capsid is selected from a different clade, such as clade A, B, C, D, or E, or from an AAV source outside of any of these clades.

rAAVhu68包括AAVhu68衣壳和载体基因组。在一个实施例中，包含rAAVhu68的组合物包含vp1的异源群体、vp2的异源群体和vp3蛋白的异源群体的组合。如本文所用，当用于指vp衣壳蛋白时，术语“异源的”或其任何语法上的变体是指由不同的元件组成的群体，例如具有不同的修饰氨基酸序列的vp1、vp2或vp3单体(蛋白质)。SEQ ID NO：21提供了AAVhu68 vp1蛋白的编码氨基酸序列。AAVhu68衣壳含有vp1蛋白、vp2蛋白和vp3蛋白内的亚群，其具有来自SEQ ID NO：21中预测的氨基酸残基的修饰。这些亚群至少包括某些脱酰胺的天冬酰胺(N或Asn)残基。例如，某些亚群在SEQ ID NO：21中的天冬酰胺-甘氨酸对中包含至少一个、两个、三个或四个高度脱酰胺的天冬酰胺(N)位置并且任选地进一步包含其它脱酰胺的氨基酸，其中脱酰胺导致氨基酸改变和其它任选的修饰。本文描述了这些和其它修饰的各种组合。rAAVhu68 comprises an AAVhu68 capsid and a vector genome. In one embodiment, a composition comprising rAAVhu68 comprises a combination of a heterologous population of vp1, a heterologous population of vp2, and a heterologous population of vp3 proteins. As used herein, the term "heterologous" or any grammatical variant thereof, when used to refer to vp capsid proteins, refers to a population composed of different elements, such as vp1, vp2, or vp3 monomers (proteins) having different modified amino acid sequences. SEQ ID NO: 21 provides the encoded amino acid sequence of the AAVhu68 vp1 protein. The AAVhu68 capsid contains subsets within the vp1 protein, the vp2 protein, and the vp3 protein that have modifications of the amino acid residues predicted from SEQ ID NO: 21. These subsets include at least some deamidated asparagine (N or Asn) residues. For example, certain subgroups comprise at least one, two, three or four highly deamidated asparagine (N) positions in the asparagine-glycine pairs in SEQ ID NO: 21 and optionally further comprise other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications. Various combinations of these and other modifications are described herein.

如本文所用，除非另有说明，否则vp蛋白的“亚群”是指具有至少一种共同定义的特征并且由至少一个组成员至少于参考组的所有成员组成的一组vp蛋白。As used herein, unless otherwise indicated, a "subgroup" of vp proteins refers to a group of vp proteins having at least one commonly defined characteristic and consisting of at least one group member that is less than all members of a reference group.

例如，除非另有说明，vp1蛋白的“亚群”是组装的AAV衣壳中的至少一(1)个vp1蛋白和少于所有的vp1蛋白。除非另有说明，vp3蛋白的“亚群”可以是组装的AAV衣壳中的一(1)个vp3蛋白至少于所有vp3蛋白。例如，vp1蛋白可以是vp蛋白的亚群；vp2蛋白可以是单独的vp蛋白亚群，而vp3是组装的AAV衣壳中的另一vp蛋白亚群。在另一实例中，vp1、vp2和vp3蛋白可以含有具有不同修饰的亚群，例如至少一个、两个、三个或四个高度脱酰胺的天冬酰胺，例如在天冬酰胺-甘氨酸对处。For example, unless otherwise specified, a "subpopulation" of vp1 proteins is at least one (1) vp1 protein and less than all vp1 proteins in an assembled AAV capsid. Unless otherwise specified, a "subpopulation" of vp3 proteins can be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid. For example, vp1 proteins can be a subpopulation of vp proteins; vp2 proteins can be a separate subpopulation of vp proteins, and vp3 is another subpopulation of vp proteins in an assembled AAV capsid. In another example, vp1, vp2, and vp3 proteins can contain subpopulations with different modifications, such as at least one, two, three, or four highly deamidated asparagines, such as at asparagine-glycine pairs.

除非另有说明，否则高度脱酰胺是指与参考氨基酸位置处的预测氨基酸序列相比，在参考氨基酸位置处至少45％脱酰胺、至少50％脱酰胺、至少60％脱酰胺、至少65％脱酰胺、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、至少99％、至多约100％脱酰胺(例如，基于总的vp1蛋白，SEQ ID NO：21的氨基酸57处的天冬酰胺的至少80％可以脱酰胺，或基于总的vp1、vp2和vp3蛋白，SEQ ID NO：21的氨基酸409处的天冬酰胺的20％可以脱酰胺)。这种百分比可以使用2D-凝胶、质谱技术或其它合适的技术来确定。Unless otherwise indicated, highly deamidated means at least 45% deamidated, at least 50% deamidated, at least 60% deamidated, at least 65% deamidated, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, up to about 100% deamidated at a reference amino acid position compared to the predicted amino acid sequence at the reference amino acid position (e.g., at least 80% of the asparagine at amino acid 57 of SEQ ID NO: 21 may be deamidated based on total vpl protein, or 20% of the asparagine at amino acid 409 of SEQ ID NO: 21 may be deamidated based on total vpl, vp2 and vp3 proteins). Such percentages may be determined using 2D-gels, mass spectrometry or other suitable techniques.

如本文提供的，SEQ ID NO：21的每个脱酰胺N可以独立地是天冬氨酸(Asp)、异天冬氨酸(isoAsp)、天冬氨酸，和/或Asp和isoAsp的互变共混物，或其组合。可以存在任何合适比率的α-和异天冬氨酸。例如，在某些实施例中，该比率可以是10∶1至1∶10天冬氨酸：异天冬氨酸，约50∶50天冬氨酸：异天冬氨酸，或约1：3天冬氨酸：异天冬氨酸，或另一选定比率。在某些实施例中，SEQ ID NO：21中的一个或多个谷氨酰胺(Q)脱酰胺为谷氨酸(Glu)，即α-谷氨酸、γ-谷氨酸(Glu)，或α-和β-谷氨酸的共混物，其可以通过常见的谷氨酰胺中间体相互转化。可以存在任何合适比率的α-和γ-谷氨酸。例如，在某些实施例中，该比率可以是从10∶1至1∶10的α∶γ，约50∶50的α∶γ，或约1∶3的α∶γ，或另一选定比率。As provided herein, each deamidated N of SEQ ID NO: 21 can independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartic acid, and/or an interconvertible blend of Asp and isoAsp, or a combination thereof. Any suitable ratio of α- and isoaspartic acid can be present. For example, in certain embodiments, the ratio can be 10:1 to 1:10 aspartic acid: isoaspartic acid, about 50:50 aspartic acid: isoaspartic acid, or about 1:3 aspartic acid: isoaspartic acid, or another selected ratio. In certain embodiments, one or more glutamines (Q) in SEQ ID NO: 21 are deamidated to glutamic acid (Glu), i.e., α-glutamic acid, γ-glutamic acid (Glu), or a blend of α- and β-glutamic acid, which can be interconverted through common glutamine intermediates. Any suitable ratio of α- and γ-glutamic acid can be present. For example, in certain embodiments, the ratio may be from 10:1 to 1:10 α:γ, approximately 50:50 α:γ, or approximately 1:3 α:γ, or another selected ratio.

因此，rAAVhu68包括rAAVhu68衣壳内具有脱酰胺氨基酸的vp1、vp2和/或vp3蛋白的亚群，至少包括至少一个含有至少一种高度脱酰胺天冬酰胺的亚群。另外，其它修饰可以包括异构化，特别是在选定的天冬氨酸(D或Asp)残基位置。在其它实施例中，修饰可以包括在Asp位置的酰胺化。Thus, rAAVhu68 includes subpopulations of vp1, vp2, and/or vp3 proteins within the rAAVhu68 capsid that have deamidated amino acids, including at least one subpopulation that contains at least one highly deamidated asparagine. Additionally, other modifications may include isomerization, particularly at selected aspartic acid (D or Asp) residue positions. In other embodiments, modifications may include amidation at the Asp position.

在某些实施例中，AAVhu68衣壳含有vp1、vp2和vp3亚群，其具有至少4至至少约25个脱酰胺氨基酸残基位置，与SEQ ID NO：21的编码氨基酸序列相比，其中至少1至10％是脱酰胺的。这些中的大部分可以是N残基。然而，Q残基也可以是脱酰胺的。In certain embodiments, the AAVhu68 capsid contains vp1, vp2, and vp3 subgroups having at least 4 to at least about 25 deamidated amino acid residue positions, of which at least 1 to 10% are deamidated compared to the amino acid sequence encoded by SEQ ID NO: 21. The majority of these can be N residues. However, Q residues can also be deamidated.

在某些实施例中，AAVhu68衣壳的特征还在于以下的一个或多个。AAVhu68衣壳蛋白，其包含：通过从编码SEQ ID NO：21的1至736的预测氨基酸序列的核酸序列表达产生的AAVhu68 vp1蛋白，由SEQ ID NO：20产生的vp1蛋白，或由与SEQ ID NO：20至少70％相同的核酸序列产生的编码SEQ ID NO：23的1至736的预测氨基酸序列的vp1蛋白；通过从编码SEQID NO：21的至少约氨基酸138至736的预测氨基酸序列的核酸序列表达产生的AAVhu68 vp2蛋白，由包含SEQ ID NO：20的至少核苷酸412至2211的序列产生的vp2蛋白，或由与SEQ IDNO：20的至少核苷酸412至2211至少70％相同的核酸序列产生的编码SEQ ID NO：21的至少约氨基酸138至736的预测氨基酸序列的vp2蛋白，和/或通过从编码SEQ ID NO：21的至少约氨基酸203至736的预测氨基酸序列的核酸序列表达产生的AAVhu68 vp3蛋白，由包含SEQID NO：20的至少核苷酸607至2211的序列产生的vp3蛋白，或由与SEQ ID NO：20的至少核苷酸607至2211至少70％相同的核酸序列产生的编码SEQ ID NO：21的至少约氨基酸203至736的预测氨基酸序列的vp3蛋白。In certain embodiments, the AAVhu68 capsid is further characterized by one or more of the following. AAVhu68 capsid protein comprising: an AAVhu68 vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:21, a vp1 protein produced from SEQ ID NO:20, or a vp1 protein encoding the predicted amino acid sequence of 1 to 736 of SEQ ID NO:23 produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO:20; an AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:21, a vp2 protein produced from a sequence comprising at least nucleotides 412 to 2211 of SEQ ID NO:20, or a vp2 protein encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:21 produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412 to 2211 of SEQ ID NO:20, and/or an AAVhu68 vp3 protein produced by expression from a nucleic acid sequence encoding at least about amino acids 138 to 736 of SEQ ID NO:21. AAVhu68 vp3 protein produced by expressing a nucleic acid sequence that is at least about the predicted amino acid sequence of amino acids 203 to 736 of SEQ ID NO:21, a vp3 protein produced by a sequence comprising at least nucleotides 607 to 2211 of SEQ ID NO:20, or a vp3 protein encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:21 produced by a nucleic acid sequence that is at least 70% identical to at least nucleotides 607 to 2211 of SEQ ID NO:20.

另外或替代地，提供了一种AAV衣壳，其包含任选地在位置157处包含缬氨酸的vp1蛋白的异源群体、任选地在位置157处包含缬氨酸的vp2蛋白的异源群体，和vp3蛋白的异源群体，其中基于SEQ ID NO：21的vp1衣壳的编号，至少vp1和vp2蛋白的亚群在位置157处包含缬氨酸并且任选地进一步在位置67处包含谷氨酸。另外或替代地，提供了一种AAVhu68衣壳，其包含：vp1蛋白的异源群体，其是编码SEQ ID NO：21的氨基酸序列的核酸序列的产物；vp2蛋白的异源群体，其是编码SEQ ID NO：21的至少约氨基酸138至736的氨基酸序列的核酸序列的产物；以及vp3蛋白的异源群体，其是编码SEQ ID NO：21的至少氨基酸203至736的核酸序列的产物，其中：vp1、vp2和vp3蛋白含有具有氨基酸修饰的亚群。Additionally or alternatively, an AAV capsid is provided that comprises a heterologous population of vp1 proteins that optionally comprise valine at position 157, a heterologous population of vp2 proteins that optionally comprise valine at position 157, and a heterologous population of vp3 proteins, wherein based on the numbering of the vp1 capsid of SEQ ID NO: 21, at least a subset of the vp1 and vp2 proteins comprise valine at position 157 and optionally further comprise glutamic acid at position 67. Additionally or alternatively, an AAVhu68 capsid is provided that comprises: a heterologous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 21; a heterologous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO: 21; and a heterologous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203 to 736 of SEQ ID NO: 21, wherein: the vp1, vp2 and vp3 proteins contain subsets having amino acid modifications.

AAVhu68 vp1、vp2和vp3蛋白通常表达为由编码SEQ ID NO：21的全长vp1氨基酸序列(氨基酸1至736)的相同核酸序列编码的替代剪接变体。任选地，单独使用vp1编码序列来表达vp1、vp2和vp3蛋白。替代地，该序列可以与编码SEQ ID NO：21的AAVhu68 vp3氨基酸序列(约aa 203至736)而不含vp1独特区(约aa 1至约aa 137)和/或vp2独特区(约aa 1至约aa202)的核酸序列或与其互补的链、相应的mRNA(SEQ ID NO：20的约nt 607至约nt 2211)或与SEQ ID NO：20至少70％至至少99％(例如，至少85％、至少90％、至少95％、至少97％、至少98％或至少99％)相同的编码SEQ ID NO：21的aa 203至736的序列中的一个或多个共表达。另外或替代地，vp1编码和/或vp2编码序列可以与编码SEQ ID NO：21的AAVhu68 vp2氨基酸序列(约aa 138至736)而不含vp1独特区(约aa 1至约137)的核酸序列或与其互补的链、相应的mRNA(SEQ ID NO：20的nt 412至2211)或与SEQ ID NO：20至少70％至至少99％(例如，至少85％、至少90％、至少95％、至少97％、至少98％或至少99％)相同的编码SEQ IDNO：21的约aa 138至736的序列共表达。AAVhu68 vp1, vp2 and vp3 proteins are typically expressed as alternative splice variants encoded by the same nucleic acid sequence encoding the full-length vp1 amino acid sequence (amino acids 1 to 736) of SEQ ID NO: 21. Optionally, the vp1 coding sequence alone is used to express the vp1, vp2 and vp3 proteins. Alternatively, the sequence can be co-expressed with a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence (about aa 203 to 736) of SEQ ID NO:21 without the vp1 unique region (about aa 1 to about aa 137) and/or the vp2 unique region (about aa 1 to about aa202), or the complementary strand thereof, the corresponding mRNA (about nt 607 to about nt 2211 of SEQ ID NO:20), or one or more of the sequence encoding aa 203 to 736 of SEQ ID NO:21 that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:20. Additionally or alternatively, the vp1-encoding and/or vp2-encoding sequences can be co-expressed with a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:21 (about aa 138 to 736) without the vp1 unique region (about aa 1 to about 137), or the complementary strand thereof, the corresponding mRNA (nt 412 to 2211 of SEQ ID NO:20), or a sequence encoding about aa 138 to 736 of SEQ ID NO:21 that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:20.

如本文所述，rAAVhu68具有在表达来自编码SEQ ID NO：21的vp1氨基酸序列的AAVhu68核酸的衣壳的生产系统中产生的rAAVhu68衣壳，以及任选的其它核酸序列，例如编码不含vp1和/或vp2独特区的vp3蛋白的核酸序列。使用单一核酸序列vp1产生的rAAVhu68产生vp1蛋白、vp2蛋白和vp3蛋白的异源群体。更具体地，rAAVhu68衣壳含有vp1蛋白、vp2蛋白和vp3蛋白内的亚群，其具有来自SEQ ID NO：21中预测的氨基酸残基的修饰。这些亚群至少包括脱酰胺的天冬酰胺(N或Asn)残基。例如，天冬酰胺-甘氨酸对中的天冬酰胺是高度脱酰胺的。As described herein, rAAVhu68 has rAAVhu68 capsids produced in a production system that expresses capsids from an AAVhu68 nucleic acid encoding the vp1 amino acid sequence of SEQ ID NO: 21, and optionally other nucleic acid sequences, such as a nucleic acid sequence encoding a vp3 protein that does not contain the unique regions of vp1 and/or vp2. rAAVhu68 produced using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1 proteins, vp2 proteins, and vp3 proteins. More specifically, rAAVhu68 capsids contain subsets within the vp1 proteins, vp2 proteins, and vp3 proteins that have modifications from the amino acid residues predicted in SEQ ID NO: 21. These subsets include at least deamidated asparagine (N or Asn) residues. For example, the asparagine in the asparagine-glycine pair is highly deamidated.

在一个实施例中，AAVhu68 vp1核酸序列具有SEQ ID NO：20的序列，或与其互补的链，例如相应的mRNA。在某些实施例中，vp2和/或vp3蛋白可以另外或替代地从与vp1不同的核酸序列表达，例如，以改变所选表达系统中vp蛋白的比率。在某些实施例中，还提供了编码SEQ ID NO：21的AAVhu68 vp3氨基酸序列(约aa 203至736)而不含vp1独特区(约aa 1至约aa 137)和/或vp2独特区(约aa 1至约aa 202)的核酸序列或与其互补的链、相应的mRNA(SEQ ID NO：20的约nt 607至约nt 2211)。在某些实施例中，还提供了编码SEQ ID NO：21的AAVhu68 vp2氨基酸序列(约aa 138至736)而不含vp1独特区(约aa 1至约137)的核酸序列或与其互补的链、相应的mRNA(SEQ ID NO：20的nt 412至2211)。In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO: 20, or a complementary strand thereof, such as a corresponding mRNA. In certain embodiments, the vp2 and/or vp3 proteins can be expressed additionally or alternatively from a different nucleic acid sequence than vp1, for example, to alter the ratio of vp proteins in a selected expression system. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 21 (about aa 203 to 736) without the vp1 unique region (about aa 1 to about aa 137) and/or the vp2 unique region (about aa 1 to about aa 202), or a complementary strand thereof, a corresponding mRNA (about nt 607 to about nt 2211 of SEQ ID NO: 20) is also provided. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 21 (about aa 138 to 736) without the vp1 unique region (about aa 1 to about 137) or a complementary strand thereof, a corresponding mRNA (nt 412 to 2211 of SEQ ID NO: 20) is also provided.

然而，可以选择编码SEQ ID NO：21的氨基酸序列的其它核酸序列用于产生rAAVhu68衣壳。在某些实施例中，核酸序列具有SEQ ID NO：20的核酸序列，或与SEQ ID NO：20至少70％至99％相同、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％或至少99％相同的编码SEQ ID NO：21的序列。在某些实施例中，核酸序列具有SEQ ID NO：20的核酸序列，或与SEQ ID NO：20的约nt 412至约nt 2211至少70％至99％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％或至少99％相同的编码SEQ ID NO：21的vp2衣壳蛋白(约aa 138至736)的序列。在某些实施例中，核酸序列具有SEQ ID NO：20的约nt607至约nt 2211的核酸序列，或与SEQ ID NO：20的约nt 607至约nt 2211至少70％至99％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％或至少99％相同的编码SEQID NO：21的vp3衣壳蛋白(约aa 203至736)的序列。However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 21 can be selected for use in generating rAAVhu68 capsids. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 20, or a sequence encoding SEQ ID NO: 21 that is at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 20. In certain embodiments, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 20, or a sequence encoding the vp2 capsid protein (about aa 138 to 736) of SEQ ID NO: 21 that is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to about nt 412 to about nt 2211 of SEQ ID NO: 20. In certain embodiments, the nucleic acid sequence has a nucleic acid sequence of about nt 607 to about nt 2211 of SEQ ID NO:20, or a sequence encoding the vp3 capsid protein (about aa 203 to 736) of SEQ ID NO:21 that is at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to about nt 607 to about nt 2211 of SEQ ID NO:20.

设计编码该rAAVhu68衣壳的核酸序列，包括DNA(基因组或cDNA)或RNA(例如，mRNA)在本领域技术范围内。在某些实施例中，编码AAVhu68 vp1衣壳蛋白的核酸序列提供于SEQ ID NO：20。在其它实施例中，可以选择与SEQ ID NO：20具有70％至99.9％同一性的核酸序列来表达AAVhu68衣壳蛋白。在某些其它实施例中，核酸序列与SEQ ID NO：20至少约75％相同、至少80％相同、至少85％、至少90％、至少95％、至少97％相同或至少99％至99.9％相同。这种核酸序列可以是密码子优化的，可以通过各种方法设计。用于在可通过各种方法设计的选定系统(即，细胞类型)中表达。这种优化可以使用在线可用的方法(绷如，GeneArt)、公开的方法或提供密码子优化服务的公司(例如，DNA2.0(加利福尼亚州门洛帕克))来进行。例如，在美国国际专利公开号WO 2015/012924中描述了一种密码子优化方法，其通过引用整体并入本文。另参见，例如，美国专利公开号2014/0032186和美国专利公开号2006/0136184。合适地，修饰用于产物的开放阅读框(ORF)的整体长度。然而，在一些实施例中，仅改变ORF的片段。通过使用这些方法之一，可以将频率应用于任何给定的多肽序列并产生编码多肽的密码子优化的编码区的核酸片段。许多选项可用于进行密码子的实际改变或用于合成如本文所述设计的密码子优化的编码区。这种修饰或合成可以使用本领域普通技术人员熟知的标准和常规分子生物学操作来进行。在一种方法中，通过标准方法合成一系列长度各为80至90个核苷酸且跨越所需序列长度的互补寡核苷酸对。合成这些寡核苷酸对，使得在退火时，它们形成含有粘性末端的80至90个碱基对的双链片段，例如，合成该对中的每个寡核苷酸以延伸3、4、5、6、7、8、9、10或更多个碱基超过与该对中的其它寡核苷酸互补的区域。每对寡核苷酸的单链端被设计成与另一对寡核苷酸的单链端退火。允许寡聚核苷酸对退火，然后允许这些双链片段中大约五至六个通过粘性单链末端退火在一起，然后将它们连接在一起并克隆到标准细菌克隆载体中，例如可以从加利福尼亚州卡尔斯巴德的英杰公司(Invitrogen Corporation)获得的载体。然后通过标准方法对构建体进行测序。制备由连接在一起的80至90个碱基对的5至6个片段，即约500个碱基对的片段组成的这些构建体中的几种，使得整个所需序列在一系列质粒构建体中表示。然后用合适的限制酶切割这些质粒的插入片段并连接在一起形成最终的构建体。然后将最终的构建体克隆到标准细菌克隆载体中，并测序。其它方法对本领域技术人员来说是显而易见的。另外，基因合成在商业上很容易获得。It is within the skill of the art to design a nucleic acid sequence encoding the rAAVhu68 capsid, including DNA (genomic or cDNA) or RNA (e.g., mRNA). In certain embodiments, the nucleic acid sequence encoding the AAVhu68 vp1 capsid protein is provided in SEQ ID NO: 20. In other embodiments, a nucleic acid sequence having 70% to 99.9% identity to SEQ ID NO: 20 can be selected to express the AAVhu68 capsid protein. In certain other embodiments, the nucleic acid sequence is at least about 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, at least 97% identical, or at least 99% to 99.9% identical to SEQ ID NO: 20. Such nucleic acid sequences can be codon optimized and can be designed by various methods. For expression in a selected system (i.e., cell type) that can be designed by various methods. Such optimization can be performed using methods available online (e.g., GeneArt), published methods, or companies that provide codon optimization services (e.g., DNA2.0 (Menlo Park, CA)). For example, a codon optimization method is described in U.S. International Patent Publication No. WO 2015/012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. Patent Publication No. 2014/0032186 and U.S. Patent Publication No. 2006/0136184. Suitably, the overall length of the open reading frame (ORF) for the product is modified. However, in some embodiments, only the fragment of the ORF is changed. By using one of these methods, the frequency can be applied to any given polypeptide sequence and produce a nucleic acid fragment of the codon-optimized coding region of the encoded polypeptide. Many options can be used to carry out the actual change of codons or for synthesizing the codon-optimized coding region designed as described herein. This modification or synthesis can be carried out using standard and conventional molecular biology operations well known to those of ordinary skill in the art. In one method, a series of complementary oligonucleotide pairs each having a length of 80 to 90 nucleotides and spanning a desired sequence length are synthesized by standard methods. The oligonucleotide pairs are synthesized so that upon annealing, they form double-stranded fragments of 80 to 90 base pairs containing sticky ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10 or more bases beyond the region complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides are designed to anneal to the single-stranded ends of the other pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal, and then approximately five to six of these double-stranded fragments are allowed to anneal together through the sticky single-stranded ends, and then they are ligated together and cloned into a standard bacterial cloning vector, such as the one available from Invitrogen Corporation of Carlsbad, California. Vector. The construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pairs, i.e., fragments of about 500 base pairs, are prepared so that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and linked together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector and sequenced. Other methods are apparent to those skilled in the art. In addition, gene synthesis is readily available commercially.

在某些实施例中，rAAVhu68 vp1、vp2和vp3蛋白中N-G对中的天冬酰胺(N)是高度脱酰胺的。在rAAVhu68衣壳蛋白的情况下，4个残基(N57、N329、N452、N512)在不同批次中通常显示脱酰胺水平＞70％，并且在大多数情况下＞90％。其它天冬酰胺残基(N94、N253、N270、N304、N409、N477和Q599)在不同批次中也显示高达约20％的脱酰胺水平。脱酰胺化水平最初使用胰蛋白酶消化鉴定并用胰凝乳蛋白酶消化验证。In certain embodiments, the asparagine (N) in the N-G pairs in the rAAVhu68 vp1, vp2, and vp3 proteins are highly deamidated. In the case of the rAAVhu68 capsid protein, four residues (N57, N329, N452, N512) typically showed deamidation levels of >70% in different batches, and >90% in most cases. Other asparagine residues (N94, N253, N270, N304, N409, N477, and Q599) also showed deamidation levels of up to about 20% in different batches. The deamidation levels were initially identified using trypsin digestion and confirmed with chymotrypsin digestion.

在某些实施例中，rAAVhu68衣壳含有AAV vp1、vp2和/或vp3衣壳蛋白的亚群，其在rAAVhu68衣壳蛋白中具有至少四个高度脱酰胺的天冬酰胺(N)位置。在某些实施例中，约20％至50％的N-N对(不包括N-N-N三联体)显示脱酰胺作用。在某些实施例中，第一个N是脱酰胺的。在某些实施例中，第二个N是脱酰胺的。在某些实施例中，脱酰胺作用在约15％至约25％的脱酰胺作用。在SEQ ID NO：21的位置259处的Q处的脱酰胺作用是AAVhu68蛋白的AAVhu68 vp1、vp2和vp3衣壳蛋白的约8％至约42％。In certain embodiments, the rAAVhu68 capsid contains a subset of AAV vp1, vp2, and/or vp3 capsid proteins that have at least four highly deamidated asparagine (N) positions in the rAAVhu68 capsid protein. In certain embodiments, about 20% to 50% of the N-N pairs (excluding N-N-N triplets) exhibit deamidation. In certain embodiments, the first N is deamidated. In certain embodiments, the second N is deamidated. In certain embodiments, the deamidation is between about 15% and about 25% deamidation. The deamidation at the Q at position 259 of SEQ ID NO: 21 is between about 8% and about 42% of the AAVhu68 vp1, vp2, and vp3 capsid proteins of the AAVhu68 protein.

在某些实施例中，rAAVhu68衣壳的特征还在于D297中vp1、vp2和vp3蛋白的酰胺化。在某些实施例中，基于SEQ ID NO：21的编号，AAVhu68衣壳中vp1、vp2和/或vp3蛋白的位置297处的D的约70％至约75％被酰胺化。在某些实施例中，衣壳的vp1、vp2和/或vp3中的至少一个Asp被异构化为D-Asp。基于SEQ ID NO：21的编号，此类异构体通常以残基位置97、107、384中的一个或多个处的Asp的小于约1％的量存在。In certain embodiments, the rAAVhu68 capsid is further characterized by amidation of the vp1, vp2, and vp3 proteins in D297. In certain embodiments, about 70% to about 75% of the D at position 297 of the vp1, vp2, and/or vp3 proteins in the AAVhu68 capsid are amidated based on the numbering of SEQ ID NO: 21. In certain embodiments, at least one Asp in vp1, vp2, and/or vp3 of the capsid is isomerized to D-Asp. Such isomers are typically present in an amount less than about 1% of the Asp at one or more of residue positions 97, 107, 384 based on the numbering of SEQ ID NO: 21.

在某些实施例中，rAAVhu68具有AAVhu68衣壳，其具有vp1、vp2和vp3蛋白，这些蛋白具有在下表中列出的位置包含一个、两个、三个、四个或更多个脱酰胺残基的组合的亚群。rAAV中的脱酰胺作用可以使用2D凝胶电泳和/或质谱和/或蛋白质建模技术来测定。在线色谱可以用Acclaim PepMap柱和Thermo UltiMate 3000RSLC系统(默飞世尔科技公司(Thermo Fisher Scientific))进行，该系统与具有NanoFlex源(赛默飞世尔科技公司)的QExactive HF耦合。使用Q Exactive HF的数据依赖性top-20方法获得MS数据，从测量扫描(200至2000m/z)中动态选择最丰富的尚未测序的前体离子。通过更高能量的碰撞解离碎裂进行测序，用预测自动增益控制确定1e5离子的目标值，用4m/z的窗口进行前体的分离。在m/z 200下以120,000的分辨率获得测量扫描。HCD谱的分辨率可以在m/z200处设置为30,000，最大离子注入时间为50ms，归一化碰撞能量为30。S透镜的RF水平设置为50，以给出由来自消化物的肽占据的m/z区域的最佳透射。可以从碎片化选择中排除具有单个、未指定或六个及更高电荷状态的前体离子。BioPharma Finder 1.0软件(赛默飞世尔科技公司)可用于分析获得的数据。对于肽图谱，使用单条目蛋白质FASTA数据库进行检索，其中氨基甲酰甲基化设置为固定修饰；并且氧化、脱酰胺和磷酸化设置为可变修饰，10-ppm的质量准确度，高蛋白酶特异性，并且MS/MS谱的置信水平为0.8。合适的蛋白酶的实例可以包括例如胰蛋白酶或胰凝乳蛋白酶。脱酰胺肽的质谱鉴定相对简单，因为脱酰胺增加了完整分子的质量+0.984Da(-OH和-NH₂基团之间的质量差)。特定肽的脱酰胺百分比通过脱酰胺肽的质量面积除以脱酰胺肽和天然肽的面积之和来确定。考虑到可能的脱酰胺位点的数目，在不同位点脱酰胺的等压物类可以在单个峰中共迁移。因此，源自具有多个潜在脱酰胺位点的肽的碎片离子可用于定位或区分多个脱酰胺位点。在这些情况下，观察到的同位素模式内的相对强度可用于特异性地确定不同脱酰胺肽异构体的相对丰度。这种方法假定所有异构物类的碎裂效率是相同的，并且独立于脱酰胺位点。本领域技术人员将理解，可以使用这些示例性方法的多种变型。例如，合适的质谱仪可以包括例如四极飞行时间质谱仪(QTOF)，如Waters Xevo或Agilent 6530，或orbitrap仪器，如Orbitrap Fusion或Orbitrap Velos(赛默飞世尔(Thermo Fisher))。合适的液相色谱系统包括例如来自沃特世(Waters)的Acquity UPLC系统或安捷伦系统(1100或1200系列)。合适的数据分析软件可以包括例如MassLynx(沃特世)、Pinpoint和Pepfinder(赛默飞世尔科技公司)、Mascot(Matrix科学公司(Matrix Science))、Peaks DB(生物信息学解决方案公司(BioinformaticsSolutions))。还可以描述其它技术，例如在X.Jin等人，《人类基因治疗方法(Hu GeneTherapy Methods)》，第28卷，第5期，第255至267页，于2017年6月16日在线发表。In certain embodiments, rAAVhu68 has an AAVhu68 capsid having vp1, vp2, and vp3 proteins having subsets comprising a combination of one, two, three, four, or more deamidated residues at the positions listed in the table below. Deamidation in rAAV can be determined using 2D gel electrophoresis and/or mass spectrometry and/or protein modeling techniques. Online chromatography can be performed using an Acclaim PepMap column and a Thermo UltiMate 3000RSLC system (Thermo Fisher Scientific) coupled to a QExactive HF with a NanoFlex source (Thermo Fisher Scientific). MS data were acquired using a data-dependent top-20 approach with the Q Exactive HF, dynamically selecting the most abundant precursor ions that have not yet been sequenced from the survey scan (200 to 2000 m/z). Sequencing was performed by higher energy collisional dissociation fragmentation, with a target value of 1e5 ions determined using predictive automatic gain control, and a window of 4 m/z was used for precursor separation. The measurement scan was obtained at a resolution of 120,000 at m/z 200. The resolution of the HCD spectrum can be set to 30,000 at m/z 200, with a maximum ion injection time of 50 ms and a normalized collision energy of 30. The RF level of the S lens is set to 50 to give the best transmission of the m/z region occupied by the peptides from the digest. Precursor ions with a single, unspecified or six and higher charge states can be excluded from the fragmentation selection. BioPharma Finder 1.0 software (Thermo Fisher Scientific) can be used to analyze the data obtained. For peptide maps, a single entry protein FASTA database is used for retrieval, wherein carbamidomethylation is set as a fixed modification; and oxidation, deamidation and phosphorylation are set as variable modifications, a mass accuracy of 10-ppm, high protease specificity, and a confidence level of 0.8 for the MS/MS spectrum. The example of a suitable protease can include, for example, trypsin or chymotrypsin. The mass spectrometric identification of deamidated peptides is relatively simple, because deamidation increases the mass of the intact molecule by +0.984Da (the mass difference between -OH and_-NH2 groups). The deamidation percentage of a particular peptide is determined by dividing the mass area of the deamidated peptide by the sum of the areas of the deamidated peptide and the native peptide. Taking into account the number of possible deamidation sites, isobaric species deamidated at different sites can co-migrate in a single peak. Therefore, fragment ions derived from peptides with multiple potential deamidation sites can be used to locate or distinguish multiple deamidation sites. In these cases, the relative intensity within the observed isotopic pattern can be used to specifically determine the relative abundance of different deamidated peptide isomers. This method assumes that the fragmentation efficiency of all isomeric species is the same and is independent of the deamidation site. It will be appreciated by those skilled in the art that a variety of variations of these exemplary methods can be used. For example, a suitable mass spectrometer can include, for example, a quadrupole time-of-flight mass spectrometer (QTOF), such as Waters Xevo or Agilent 6530, or an orbitrap instrument, such as Orbitrap Fusion or Orbitrap Velos (Thermo Fisher). Suitable liquid chromatography systems include, for example, Acquity UPLC systems or Agilent systems (1100 or 1200 series) from Waters. Suitable data analysis software may include, for example, MassLynx (Waters), Pinpoint and Pepfinder (Thermo Fisher Scientific), Mascot (Matrix Science), Peaks DB (Bioinformatics Solutions). Other techniques may also be described, for example in X. Jin et al., "Human Gene Therapy Methods", Vol. 28, No. 5, pp. 255 to 267, published online on June 16, 2017.

在某些实施例中，AAVhu68衣壳的特征在于具有衣壳蛋白，其中基于SEQ ID NO：21的氨基酸序列编号，至少45％的N残基在位置N57、N329、N452和/或N512中的至少一个被脱酰胺。在某些实施例中，在这些N-G位置(即，N57、N329、N452和/或N512，基于SEQ ID NO：21的氨基酸序列编号)的一个或多个处的N残基的至少约60％、至少约70％、至少约80％或至少90％被脱酰胺。在这些和其它实施例中，AAVhu68衣壳的特征还在于具有这样的蛋白质群体，其中约1％至约20％的N残基在以下的一个或多个位置具有脱酰胺作用：N94、N253、N270、N304、N409、N477和/或Q599，基于SEQ ID NO：21的氨基酸序列编号。在某些实施例中，AAVhu68包含vp1、vp2和/或vp3蛋白的至少一个亚群，其在基于SEQ ID NO：21的氨基酸序列编号的位置N35、N57、N66、N94、N113、N252、N253、Q259、N270、N303、N304、N305、N319、N328、N329、N336、N409、N410、N452、N477、N515、N598、Q599、N628、N651、N663、N709、N735中的一个或多个位置处脱酰胺，或其组合。在某些实施例中，衣壳蛋白可以具有一个或多个酰胺化的氨基酸。In certain embodiments, the AAVhu68 capsid is characterized as having a capsid protein in which at least 45% of the N residues are deamidated at at least one of positions N57, N329, N452, and/or N512, based on the amino acid sequence numbering of SEQ ID NO: 21. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, or at least 90% of the N residues at one or more of these N-G positions (i.e., N57, N329, N452, and/or N512, based on the amino acid sequence numbering of SEQ ID NO: 21) are deamidated. In these and other embodiments, the AAVhu68 capsid is further characterized as having a population of proteins in which about 1% to about 20% of the N residues have deamidation at one or more of the following positions: N94, N253, N270, N304, N409, N477, and/or Q599, based on the amino acid sequence numbering of SEQ ID NO: 21. In certain embodiments, AAVhu68 comprises at least a subset of vp1, vp2, and/or vp3 proteins that are deamidated at one or more of positions N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, or a combination thereof, based on the amino acid sequence numbering of SEQ ID NO: 21. In certain embodiments, the capsid protein may have one or more amidated amino acids.

还观察到其它修饰，其中大多数不导致一个氨基酸转化为不同的氨基酸残基。任选地，衣壳的vp1、vp2和vp3中的至少一个Lys被乙酰化。任选地，衣壳的vp1、vp2和/或vp3中的至少一个Asp被异构化为D-Asp。任选地，衣壳的vp1、vp2和/或vp3中的至少一个S(Ser，丝氨酸)被磷酸化。任选地，衣壳的vp1、vp2和/或vp3中的至少一个T(Thr，苏氨酸)被磷酸化。任选地，衣壳的vp1、vp2和/或vp3中的至少一个W(trp，色氨酸)被氧化。任选地，衣壳的vp1、vp2和/或vp3中的至少一个M(Met，甲硫氨酸)被氧化。在某些实施例中，衣壳蛋白具有一个或多个磷酸化。例如，某些vp1衣壳蛋白可以在位置149被磷酸化。Other modifications are also observed, most of which do not result in the conversion of one amino acid into a different amino acid residue. Optionally, at least one Lys in vp1, vp2 and vp3 of the capsid is acetylated. Optionally, at least one Asp in vp1, vp2 and/or vp3 of the capsid is isomerized to D-Asp. Optionally, at least one S (Ser, serine) in vp1, vp2 and/or vp3 of the capsid is phosphorylated. Optionally, at least one T (Thr, threonine) in vp1, vp2 and/or vp3 of the capsid is phosphorylated. Optionally, at least one W (trp, tryptophan) in vp1, vp2 and/or vp3 of the capsid is oxidized. Optionally, at least one M (Met, methionine) in vp1, vp2 and/or vp3 of the capsid is oxidized. In certain embodiments, the capsid protein has one or more phosphorylations. For example, some vp1 capsid proteins can be phosphorylated at position 149.

在某些实施例中，rAAVhu68衣壳包含vp1蛋白的异源群体，vp1蛋白是编码SEQ IDNO：21的氨基酸序列的核酸序列的产物，其中vp1蛋白包含在位置67处的谷氨酸(Glu)和/或在位置157处的缬氨酸(Val)；任选地在位置157处包含缬氨酸(Val)的vp2蛋白的异源群体；以及vp3蛋白的异源群体。AAVhu68衣壳含有至少一个亚群，其中基于SEQ ID NO：21的氨基酸序列的残基编号，位于vp1蛋白的位置57处的天冬酰胺-甘氨酸对中的至少65％的天冬酰胺(N)和位于vp1、v2和vp3蛋白的位置329、452和/或512处的天冬酰胺-甘氨酸对中的至少70％的天冬酰胺(N)被脱酰胺，其中脱酰胺作用导致氨基酸改变。如本文更详细讨论的，脱酰胺天冬酰胺可以脱酰胺为天冬氨酸、异天冬氨酸、相互转化的天冬氨酸/异天冬氨酸对或其组合。在某些实施例中，rAAVhu68的特征还在于以下一个或多个：(a)vp2蛋白中的每一种独立地是至少编码SEQ ID NO：21的vp2蛋白的核酸序列的产物；(b)vp3蛋白中的每一种独立地是至少编码SEQ ID NO：21的vp3蛋白的核酸序列的产物；(c)编码vp1蛋白的核酸序列是SEQ ID NO：21，或与SEQ ID NO：20至少70％至至少99％(例如，至少85％、至少90％、至少95％、至少97％、至少98％或至少99％)相同的编码SEQ ID NO：21的氨基酸序列的序列。任选地，该序列单独用于表达vp1、vp2和vp3蛋白。替代地，该序列可以与编码SEQ ID NO：21的AAVhu68 vp3氨基酸序列(约aa 203至736)而不含vp1独特区(约aa 1至约aa 137)和/或vp2独特区(约aa 1至约aa 202)的核酸序列或与其互补的链、相应的mRNA(SEQ ID NO：20的约nt 607至约nt 2211)或与SEQ ID NO：20至少70％至至少99％(例如，至少85％、至少90％、至少95％、至少97％、至少98％或至少99％)相同的编码SEQ ID NO：21的aa 203至736的序列中的一个或多个共表达。另外或替代地，vp1编码和/或vp2编码序列可以与编码SEQ IDNO：21的AAVhu68 vp2氨基酸序列(约aa 138至736)而不含vp1独特区(约aa 1至约137)的核酸序列或与其互补的链、相应的mRNA(SEQ ID NO：20的nt 412至2211)或与SEQ ID NO：20至少70％至至少99％(例如，至少85％、至少90％、至少95％、至少97％、至少98％或至少99％)相同的编码SEQ lD NO：21的约aa 138至736的序列共表达。In certain embodiments, the rAAVhu68 capsid comprises a heterogeneous population of vp1 proteins, which are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 21, wherein the vp1 proteins comprise glutamic acid (Glu) at position 67 and/or valine (Val) at position 157; a heterogeneous population of vp2 proteins, optionally comprising valine (Val) at position 157; and a heterogeneous population of vp3 proteins. The AAVhu68 capsid contains at least one subpopulation, wherein at least 65% of the asparagines (N) in the asparagine-glycine pairs at position 57 of the vp1 protein and at least 70% of the asparagines (N) in the asparagine-glycine pairs at positions 329, 452, and/or 512 of the vp1, v2, and vp3 proteins are deamidated based on residue numbering of the amino acid sequence of SEQ ID NO: 21, wherein the deamidation results in an amino acid change. As discussed in more detail herein, the deamidated asparagine can be deamidated to aspartic acid, isoaspartic acid, an interconverted aspartic acid/isoaspartic acid pair, or a combination thereof. In certain embodiments, rAAVhu68 is further characterized by one or more of the following: (a) each of the vp2 proteins is independently the product of a nucleic acid sequence encoding at least the vp2 protein of SEQ ID NO: 21; (b) each of the vp3 proteins is independently the product of a nucleic acid sequence encoding at least the vp3 protein of SEQ ID NO: 21; (c) the nucleic acid sequence encoding the vp1 protein is SEQ ID NO: 21, or a sequence encoding the amino acid sequence of SEQ ID NO: 21 that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 20. Optionally, the sequence is used alone to express the vp1, vp2, and vp3 proteins. Alternatively, the sequence can be co-expressed with a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence (about aa 203 to 736) of SEQ ID NO:21 without the vp1 unique region (about aa 1 to about aa 137) and/or the vp2 unique region (about aa 1 to about aa 202), or the complementary strand thereof, the corresponding mRNA (about nt 607 to about nt 2211 of SEQ ID NO:20), or one or more of the sequence encoding aa 203 to 736 of SEQ ID NO:21 that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:20. Additionally or alternatively, the vp1-encoding and/or vp2-encoding sequences can be co-expressed with a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:21 (approximately aa 138 to 736) without the vp1 unique region (approximately aa 1 to approximately 137), or the complementary strand thereof, the corresponding mRNA (nt 412 to 2211 of SEQ ID NO:20), or a sequence encoding approximately aa 138 to 736 of SEQ ID NO:21 that is at least 70% to at least 99% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:20.

另外或替代地，rAAVhu68衣壳包含vp1、vp2和/或vp3蛋白的至少一个亚群，其在基SEQ ID NO：21的编号的位置N57、N66、N94、N113、N252、N253、Q259、N270、N303、N304、N305、N319、N328、N329、N336、N409、N410、N452、N477、N512、N515、N598、Q599、N628、N651、N663、N709中的一个或多个位置处脱酰胺，或其组合；(e)rAAVhu68衣壳包含vp1、vp2和/或vp3蛋白的亚群，其包含基于SEQ ID NO：21的编号在位置N66、N94、N113、N252、N253、Q259、N270、N303、N304、N305、N319、N328、N336、N409、N410、N477、N515、N598、Q599、N628、N651、N663、N709中的一个或多个位置处的1％至20％脱酰胺，或其组合；(f)rAAVhu68衣壳包含vp1亚群，其中基于SEQ ID NO：21的编号，在vpl蛋白的位置57处的65％至100％的N被脱酰胺；(g)rAAVhu68衣壳包含vp1蛋白的亚群，其中vp1蛋白的位置57处的N的75％至100％被脱酰胺；(h)rAAVhu68衣壳包含vp1蛋白、vp2蛋白和/或vp3蛋白的亚群，其中基于SEQ ID NO：21的编号，在位置329处的N的80％至100％被脱酰胺；(i)rAAVhu68衣壳包含vp1蛋白、vp2蛋白和/或vp3蛋白的亚群，其中基于SEQ ID NO：21的编号，在位置452处的N的80％至100％被脱酰胺；(j)rAAVhu68衣壳包含vp1蛋白、vp2蛋白和/或vp3蛋白的亚群，其中基于SEQ ID NO：21的编号，在位置512处的N的80％至100％被脱酰胺；(k)rAAV包含约60种总衣壳蛋白，比例为约1vp1蛋白：约1至1.5vp2蛋白：3至10vp3蛋白；(1)rAAV包含约60种总衣壳蛋白，比例为约lvp1蛋白：约1vp2蛋白：3至9vp3蛋白。Additionally or alternatively, the rAAVhu68 capsid comprises at least one subset of vp1, vp2 and/or vp3 proteins that are deamidated at one or more of positions N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N512, N515, N598, Q599, N628, N651, N663, N709, or a combination thereof based on the numbering of SEQ ID NO: 21; (e) the rAAVhu68 capsid comprises at least one subset of vp1, vp2 and/or vp3 proteins that are deamidated at one or more of positions N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N512, N515, N598, Q599, N628, N651, N663, N709, or a combination thereof based on the numbering of SEQ ID NO: NO: 21 has 1% to 20% deamidation at one or more of positions N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N336, N409, N410, N477, N515, N598, Q599, N628, N651, N663, N709, or a combination thereof; (f) the rAAVhu68 capsid comprises the vp1 subgroup, wherein based on SEQ ID (g) rAAVhu68 capsids comprise a subset of vp1 proteins wherein 75% to 100% of the N at position 57 of the vp1 proteins are deamidated based on the numbering of SEQ ID NO:21; (h) rAAVhu68 capsids comprise a subset of vp1 proteins, vp2 proteins, and/or vp3 proteins wherein 80% to 100% of the N at position 329 are deamidated based on the numbering of SEQ ID NO:21; (i) rAAVhu68 capsids comprise a subset of vp1 proteins, vp2 proteins, and/or vp3 proteins wherein 80% to 100% of the N at position 452 are deamidated based on the numbering of SEQ ID NO:21; (j) rAAVhu68 capsids comprise a subset of vp1 proteins, vp2 proteins, and/or vp3 proteins wherein 80% to 100% of the N at position 452 are deamidated based on the numbering of SEQ ID NO:21. NO: 21, 80% to 100% of the N at position 512 is deamidated; (k) rAAV contains about 60 total capsid proteins, with a ratio of about 1 vp1 protein: about 1 to 1.5 vp2 proteins: 3 to 10 vp3 proteins; (1) rAAV contains about 60 total capsid proteins, with a ratio of about lvp1 protein: about 1 vp2 protein: 3 to 9 vp3 proteins.

在某些实施例中，修饰AAVhu68以改变天冬酰胺-甘氨酸对中的甘氨酸，以减少脱酰胺作用。在其它实施例中，天冬酰胺被改变为不同的氨基酸，例如以较慢的速率脱酰胺的谷氨酰胺；或缺少酰胺基的氨基酸(例如，谷氨酰胺和天冬酰胺含有酰胺基)；和/或缺少胺基的氨基酸(例如，赖氨酸、精氨酸和组氨酸含有酰胺基)。如本文所用，缺少酰胺或胺侧基的氨基酸是指例如甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、苏氨酸、胱氨酸、苯内氨酸、酪氨酸或色氨酸和/或脯氨酸。如所述的修饰可以是在编码的AAVhu68氨基酸序列中发现的一个、两个或三个天冬酰胺-甘氨酸对。在某些实施例中，不对所有四种天冬酰胺-甘氨酸对进行此类修饰。因此，提供了一种用于减少具有较低脱酰胺率的rAAVhu68和/或工程化rAAVhu68变体的脱酰胺作用的方法。另外，可以将一个或多个其它酰胺氨基酸改变为非酰胺氨基酸以减少rAAVhu68的脱酰胺作用。In certain embodiments, AAVhu68 is modified to change the glycine in the asparagine-glycine pair to reduce deamidation. In other embodiments, asparagine is changed to a different amino acid, such as glutamine, which deamidates at a slower rate; or an amino acid lacking an amide group (e.g., glutamine and asparagine contain an amide group); and/or an amino acid lacking an amine group (e.g., lysine, arginine, and histidine contain an amide group). As used herein, an amino acid lacking an amide or amine side group refers to, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cystine, phenylalanine, tyrosine, or tryptophan and/or proline. The modification as described can be one, two, or three asparagine-glycine pairs found in the encoded AAVhu68 amino acid sequence. In certain embodiments, such modifications are not performed on all four asparagine-glycine pairs. Therefore, a method for reducing deamidation of rAAVhu68 and/or engineered rAAVhu68 variants having a lower deamidation rate is provided. Additionally, one or more of the other amide amino acids may be changed to non-amide amino acids to reduce deamidation of rAAVhu68.

这些氨基酸修饰可以通过常规基因工程技术进行。例如，可以生成含有修饰的AAVhu68 vp密码子的核酸序列，其中编码SEQ ID NO：21(天冬酰胺-甘氨酸对)中位置58、330、453和/或513处甘氨酸的一至三个密码子被修饰以编码除甘氨酸以外的氨基酸。在某些实施例中，含有修饰的天冬酰胺密码子的核酸序列可以在位于SEQ ID NO：21中位置57、329、452和/或512处的天冬酰胺-甘氨酸对中的一至三个处被工程化，使得修饰的密码子编码除天冬酰胺以外的氨基酸。每个修饰的密码子可以编码不同的氨基酸。替代地，一个或多个改变的密码子可以编码相同的氨基酸。在某些实施例中，这些修饰的AAVhu68核酸序列可用于生成突变体rAAVhu68，其衣壳的脱酰胺作用低于天然hu68衣壳。这种突变体rAAVhu68可以具有降低的免疫原性和/或增加的储存稳定性，特别是以悬浮液形式储存。如本文所用，“密码子”是指编码氨基酸的序列中的三个核苷酸。These amino acid modifications can be made by conventional genetic engineering techniques. For example, a nucleic acid sequence containing a modified AAVhu68 vp codon can be generated, wherein one to three codons encoding glycine at positions 58, 330, 453 and/or 513 in SEQ ID NO: 21 (asparagine-glycine pairs) are modified to encode an amino acid other than glycine. In certain embodiments, a nucleic acid sequence containing a modified asparagine codon can be engineered at one to three of the asparagine-glycine pairs at positions 57, 329, 452 and/or 512 in SEQ ID NO: 21, such that the modified codon encodes an amino acid other than asparagine. Each modified codon can encode a different amino acid. Alternatively, one or more altered codons can encode the same amino acid. In certain embodiments, these modified AAVhu68 nucleic acid sequences can be used to generate mutant rAAVhu68, whose capsid has less deamidation than the native hu68 capsid. Such mutant rAAVhu68 can have reduced immunogenicity and/or increased storage stability, particularly when stored in a suspension. As used herein, a "codon" refers to three nucleotides in a sequence that encodes an amino acid.

如本文所用，“编码的氨基酸序列”是指基于被翻译为氨基酸的参考核酸序列的已知DNA密码子的翻译而预测的氨基酸。下表说明了DNA密码子和20种常见氨基酸，示出了单字母代码(SLC)和三字母代码(3LC)。As used herein, "encoded amino acid sequence" refers to the predicted amino acid based on translation of known DNA codons of a reference nucleic acid sequence translated into amino acids. The following table illustrates DNA codons and 20 common amino acids, showing single letter codes (SLC) and three letter codes (3LC).

如本文所用，术语“进化枝”当涉及AAV组时是指基于AAV vp1氨基酸序列的比对，使用邻接算法通过至少75％(至少1000个复制品)的自举值和不超过0.05的泊松校正距离测量值确定的一组彼此系统发育相关的AAV。在文献中已经描述了邻接算法。参见，例如，M.Nei和S.Kumar，《分子进化与系统发育(Molecular Evolution and Phylogenetics)》(牛津大学山版社(Oxford UniversityPress)，纽约(2000)。可以使用计算机程序来实现这种算法。例如，MEGAv2.1程序实施改进的Nei-Gojobori方法。使用这些技术和计算机程序，以及AAVvp1衣壳蛋白的序列，本领域技术人员可以容易地确定所选择的AAV是否包含在本文鉴定的一个进化枝中，在另一个进化枝中，或在这些进化枝之外。参见，例如，G Gao等人，《病毒学杂志(J Virol)》，2004年6月；78(10：6381-6388，其鉴定进化枝A、B、C、D、E和F，基因库入藏号AY530553至AY530629。另参见，WO 2005/033321。As used herein, the term "clade" when referring to an AAV group refers to a group of AAVs that are phylogenetically related to each other based on an alignment of AAV vp1 amino acid sequences using a neighbor-joining algorithm with a bootstrap value of at least 75% (at least 1000 replicates) and a Poisson-corrected distance measurement of no more than 0.05. Neighbor-joining algorithms have been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Such algorithms can be implemented using computer programs. For example, the MEGAv2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of the AAVvp1 capsid protein, one skilled in the art can readily determine whether a selected AAV is contained in one clade identified herein, in another clade, or outside of these clades. See, e.g., G Gao et al., J Virol, 2004 Jun; 78(10:6381-6388, which identifies clades A, B, C, D, E, and F, GenBank Accession Nos. AY530553 to AY530629. See also, WO 2005/033321.

已经描述了生成衣壳及其编码序列的方法，以及生产rAAV病毒载体的方法。参，见，例如，Gao等人，《美国国家科学院院刊》100(10)，6081-6086(2003)和US 2013/0045186A1。Methods for generating capsids and their coding sequences, as well as methods for producing rAAV viral vectors, have been described. See, for example, Gao et al., Proceedings of the National Academy of Sciences of the United States of America 100(10), 6081-6086 (2003) and US 2013/0045186A1.

ITR或其它AAV组分可以使用本领域技术人员可获得的技术从AAV容易地分离或工程化。这种AAV可以是分离的、工程化的或从学术，商业或公共来源(例如，美国菌种保藏中心，弗吉尼亚州马纳萨斯(American Type Culture Collection，Manassas，VA))获得。替代地，AAV序列可以通过合成或其它合适的方法通过参考已公开的序列而被工程化，这些公开的序列如文献中或数据库(例如，基因库(GenBank)、PubMed等)中可获得的。AAV病毒可以通过常规分子生物学技术工程化，使得可以优化这些颗粒用于核酸序列的细胞特异性递送，用于最小化免疫原性，用于调整稳定性和颗粒寿命，用于有效降解，用于精确递送至细胞核等。ITRs or other AAV components can be easily isolated or engineered from AAV using techniques available to those skilled in the art. Such AAV can be isolated, engineered or obtained from academic, commercial or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences can be engineered by synthesis or other suitable methods by reference to published sequences, such as those available in the literature or in databases (e.g., GenBank, PubMed, etc.). AAV viruses can be engineered by conventional molecular biology techniques so that these particles can be optimized for cell-specific delivery of nucleic acid sequences, for minimizing immunogenicity, for adjusting stability and particle life, for efficient degradation, for precise delivery to the nucleus, etc.

在某些实施例中，rAAV是自互补的AAV。“自互补的AAV”是指其中重组AAV核酸序列携带的编码区已被设计形成分子内双链DNA模板的构建体。在感染时，scAAV的两个互补半部分将结合形成一个双链DNA(dsDNA)单元，准备立即复制和转录，而不是等待细胞介导的第二条链的合成。参见，例如，D M McCarty等人，《自互补重组腺相关病毒(scAAV)载体促进独立于DNA合成的有效转导(Self-complementary recombinant adeno-associated virus(scAAV)vectors promote efficient transduction independently of DNAsynthesis)》，《基因治疗》(2001年8月)，第8卷，第16期，第1248至1254页。自互补的AAV描述于例如美国专利第6,596,535号；第7,125,717号；和第7,456,683号，其各自以全文引用的方式并入本文。In certain embodiments, rAAV is a self-complementary AAV. "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. During infection, the two complementary halves of scAAV will combine to form a double-stranded DNA (dsDNA) unit, ready to replicate and transcribe immediately, rather than waiting for the synthesis of the second chain mediated by the cell. See, for example, D M McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", "Gene Therapy" (August 2001), Vol. 8, No. 16, pp. 1248 to 1254. Self-complementary AAVs are described, e.g., in U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

在某些实施例中，rAAV是抗核酸酶的。这种核酸酶可以是单一核酸酶或核酸酶的混合物，并且可以是核酸内切酶或核酸外切酶。抗核酸酶的rAAV表明AAV衣壳已完全组装并保护这些包装的基因组序列在核酸酶孵育步骤期间免于降解(消化)，核酸酶孵育步骤被设计用于除去可能存在于生产过程中的污染核酸。在许多情况下，本文所述的rAAV是DNase抗性的。In certain embodiments, rAAV is resistant to nuclease. This nuclease can be a single nuclease or a mixture of nucleases, and can be an endonuclease or an exonuclease. Nuclease-resistant rAAV indicates that the AAV capsid has been fully assembled and protects the packaged genomic sequences from degradation (digestion) during the nuclease incubation step, and the nuclease incubation step is designed to remove contaminating nucleic acids that may be present in the production process. In many cases, the rAAV described herein is DNase resistant.

本文所述的重组腺相关病毒(AAV)可以使用已知的技术生成。参见，例如，WO2003/042397；WO 2005/033321、WO 2006/110689；US 7588772 B2。这种方法涉及培养含有编码AAV衣壳的核酸序列的宿主细胞；功能性rep基因；如本文所述的表达盒，其侧翼为AAV反向末端重复序列(ITR)；和足够的辅助功能以允许表达盒包装到AAV衣壳蛋白中。本文还提供了含有编码AAV衣壳的核酸序列的宿主细胞；功能性rep基因；所述载体基因组；和足够的辅助功能以允许将载体基因组包装到AAV衣壳蛋白中。在一个实施例中，宿主细胞是HEK293细胞。这些方法在WO2017160360 A2中有更详细的描述，其通过引用并入本文。The recombinant adeno-associated virus (AAV) described herein can be generated using known techniques. See, for example, WO2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2. This method involves culturing a host cell containing a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein, flanked by an AAV inverted terminal repeat sequence (ITR); and sufficient auxiliary functions to allow the expression cassette to be packaged into the AAV capsid protein. Also provided herein is a host cell containing a nucleic acid sequence encoding an AAV capsid; a functional rep gene; the vector genome; and sufficient auxiliary functions to allow the vector genome to be packaged into the AAV capsid protein. In one embodiment, the host cell is a HEK293 cell. These methods are described in more detail in WO2017160360 A2, which is incorporated herein by reference.

可以使用本领域技术人员可获得的产生rAAV的其它方法。合适的方法可以包括但不限于杆状病毒表达系统或通过酵母的生产。参见，例如，Robert M.Kotin，《大规模重组腺相关病毒生产(Large-scale recombinant adeno-associated virus production)》《人类分子遗传学(Hum Mol Genet.)》2011年4月15日；20(R1)：R2-R6。于2011年4月29日在线发表。doi：10.1093/hmg/ddr141；Aucoin MG等人，《利用三重感染在昆虫细胞中生产腺相关病毒载体：杆状病毒浓度比的优化(Production of adeno-associated viral vectors ininsect cells using triple infection：optimization of baculovirus concentrationratios)》《生物技术与生物工程(Biotechnol Bioeng.)》2006年12月20日；95(6)：1081-92；SAMI S.THAKUR，《在酵母中生产重组腺相关病毒载体(Production of RecombinantAdeno-associated viral vectors in yeast)》提交给佛罗里达大学研究生院的论文，2012；Kondratov O等人《在人与昆虫细胞中制备的重组腺相关病毒载体的直接头对头评估(Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral VectorsManufactured in Human versus Insect Cells)》，《分子疗法》2017年8月10日。pii：S1525-0016(17)30362-3。doi：10.1016/j.ymthe.2017.08.003.[印刷出版前的电子版]；Mietzsch M等人，OneBac 2.0：《用于生产AAV1、AAV2和AAV8载体的Sf9细胞系，具有最小的外源DNA包被(Sf9Cell Lines for Production of AAV1，AAV2，and AAV8 Vectors withMinimal Encapsidation of Foreign DNA)》《人类基因治疗方法(Hum GeneTherMethods)》2017年2月；28(1)：15-22.doi：10.1089/hgtb.2016.164；Li L等人《新型重组腺相关病毒复制型基因组的产生和表征：用于基因转移的真核DNA来源(Production andcharacterization ofnovel recombinant adeno-associated virus replicative-formgenomes：a eukaryotic source of DNA for gene transfer)》《美国科学公共图书馆(PLoS One.)》2013年8月1日；8(8)：e69879.doi：10.1371/journal.pone.0069879.2013印本；Galibert等人，《昆虫细胞中腺相关病毒载体大规模生产对治疗神经肌肉疾病的最新进展(Latest developments in the large-scale production of adeno-associatedvirus vectors in insect cells toward the treatment of neuromusculardiseases)》《无脊椎动物病理学杂志(J Invertebr Pathol.)》2011年7月；107补编：S80-93。doi：10.1016/j.jip.2011.05.008；和Kotin RM，《大规模重组腺相关病毒生产(Large-scale recombinant adeno-associated virus production)》《人类分子遗传学(Hum MolGenet.)》2011年4月15日；20(R1)：R2-6.doi：10.1093/hmg/ddr141。电子版2011年4月29日。Other methods of producing rAAV available to those skilled in the art may be used. Suitable methods may include, but are not limited to, baculovirus expression systems or production by yeast. See, e.g., Robert M. Kotin, Large-scale recombinant adeno-associated virus production, Hum Mol Genet., April 15, 2011; 20(R1): R2-R6. Published online April 29, 2011. doi: 10.1093/hmg/ddr141; Aucoin MG et al., Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios. Biotechnol Bioeng. 2006 Dec 20;95(6):1081-92; Sami S. Thakur, Production of Recombinant Adeno-associated viral vectors in yeast. Paper submitted to the Graduate School of the University of Florida, 2012; Kondratov O et al., Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells. Cells. Molecular Therapy. August 10, 2017. pii: S1525-0016(17)30362-3. doi: 10.1016/j.ymthe.2017.08.003. [Epub ahead of print]; Mietzsch M et al. OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA. Hum Gene Ther Methods. 2017 Feb;28(1):15-22. doi: 10.1089/hgtb.2016.164; Li L et al. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer. transfer. PLoS One. 2013 Aug 1;8(8):e69879. doi:10.1371/journal.pone.0069879.2013. Print. Galibert et al. Latest developments in the large-scale production of adeno-associatedvirus vectors in insect cells toward the treatment of neuromuscular diseases. J Invertebr Pathol. 2011 Jul;107 Suppl:S80-93. doi:10.1016/j.jip.2011.05.008; and Kotin RM. Large-scale recombinant adeno-associated virus production. Hum Mol Genet. 2011 Apr 15;20(R1):R2-6. doi:10.1093/hmg/ddr141. Epub 2011 Apr 29.

本领域已知多种AAV纯化方法。参见例如题为《AAV9的可规模化纯化方法(Scalable Purification Method for AAV9)》的WO 2017/160360，其通过引用并入本文，并描述了通常用于进化枝F衣壳的方法。使用两步亲和色谱法纯化，接着阴离子交换树脂色谱法纯化载体药物产物并除去空衣壳。粗细胞采集物可以经历以下步骤，如载体采集物的浓缩、载体采集物的渗滤、载体采集物的微流化、载体采集物的核酸酶消化、微流化中间体的过滤、通过色谱法的粗纯化、通过超速离心的粗纯化、通过切向流过滤的缓冲液交换，和/或配制和过滤以制备批量载体。在阴离子交换树脂层析之后进行亲和层析纯化用于纯化载体药物产物和除去空衣壳。在一个实例中，对于亲和层析步骤，渗滤产物可应用于有效捕获AAV2/9血清型的Capture Select^TM Poros-AAV2/9亲和树脂(生命科技公司(LifeTechnologies))。在这些离子条件下，显著百分比的残留细胞DNA和蛋白质流经色谱柱，而AAV颗粒被有效捕获。另参见，WO2021/158915；WO2019/241535；和WO 2021/165537。A variety of AAV purification methods are known in the art. See, for example, WO 2017/160360 entitled "Scalable Purification Method for AAV9", which is incorporated herein by reference, and describes a method commonly used for clade F capsids. Two-step affinity chromatography purification is used, followed by anion exchange resin chromatography to purify the carrier drug product and remove empty capsids. The crude cell collection can undergo the following steps, such as concentration of the carrier collection, diafiltration of the carrier collection, microfluidization of the carrier collection, nuclease digestion of the carrier collection, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and/or preparation and filtration to prepare batch carriers. Affinity chromatography purification is performed after anion exchange resin chromatography for purifying the carrier drug product and removing empty capsids. In one example, for the affinity chromatography step, the diafiltration product can be applied to Capture Select^™ Poros-AAV2/9 affinity resin (Life Technologies) that effectively captures the AAV2/9 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and protein flowed through the chromatographic column, while AAV particles were effectively captured. See also, WO2021/158915; WO2019/241535; and WO 2021/165537.

本领域技术人员可获得表征或定量rAAV的常规方法。为了计算空的和满的颗粒含量，将选定样品(例如，在本文的实例中，碘克沙醇梯度纯化的制剂，其中GC的数量＝颗粒的数量)的VP3谱带体积相对于附着的GC颗粒作图。所得线性方程(y＝mx+c)用于计算供试品峰的谱带体积中的颗粒数。然后将每20μL负载的颗粒数(pt)乘以50，得到颗粒(pt)/mL。Pt/mL除以GC/mL得到颗粒与基因组拷贝的比率(pt/GC)。Pt/mL-GC/mL得到空pt/mL。空pt/mL除以pt/mL，x100得到空颗粒的百分比。通常，用包装的基因组测定空衣壳和AAV载体颗粒的方法是本领域已知的。参见，例如，Grimm等人，《基因治疗》(1999)6：1322-1330；Sommer等人，《分子疗法(Molec.Ther.)》(2003)7：122-128。为了测试变性的衣壳，该方法包括将处理的AAV原种进行SDS-聚丙烯酰胺凝胶电泳，其由能够分离三种衣壳蛋白的任何凝胶组成，例如，在缓冲液中含有3至8％Tris-乙酸酯的梯度凝胶，然后运行凝胶直到样品材料被分离，并将凝胶印迹到尼龙或硝化纤维膜上，优选地尼龙。然后将抗AAV衣壳抗体用作与变性衣壳蛋白结合的第一抗体，优选地抗AAV衣壳单克隆抗体，最优选地B1抗AAV-2单克隆抗体(Wobus等人，《病毒学杂志》(2000)74：9281-9293)。然后使用第二抗体，其与第一抗体结合并含有用于检测与第一抗体结合的手段，更优选地含有与其共价结合的检测分子的抗IgG抗体，最优选地与辣根过氧化物酶共价连接的羊抗小鼠IgG抗体。用于检测结合的方法用于半定量测定第一抗体和第二抗体之间的结合，优选地能够检测放射性同位素发射、电磁辐射或比色变化的检测方法，最优选地化学发光检测试剂盒。例如，对于SDS-PAGE，可以从柱级分中取出样品并在含有还原剂(例如，DTT)的SDS-PAGE上样缓冲液中加热，并且将衣壳蛋白在预制梯度聚丙烯酰胺凝胶(例如，Novex)上解析。可以使用SilverXpress(英杰公司(Invitrogen)，加利福尼亚州)根据制造商的说明书或其它合适的染色方法(即SYPRO红宝石或考马斯染色)进行银染色。在一个实施例中，可以通过定量实时PCR(Q-PCR)测量柱级分中AAV载体基因组(vg)的浓度。将样品稀释并用DNase I(或另一种合适的核酸酶)消化以除去外源DNA。在核酸酶失活后，使用引物和对引物之间的DNA序列特异性的TaqMan^TM荧光探针进一步稀释和扩增样品。在Applied Biosystems Prism 7700序列检测系统上测量每个样品达到规定荧光水平所需的循环数(阈值循环，Ct)。使用含有与AAV载体中所含序列相同的序列的质粒DNA在Q-PCR反应中生成标准曲线。从样品获得的循环阈值(Ct)值用于通过将其标准化为质粒标准曲线的Ct值来确定载体基因组滴度。也可以使用基于数字PCR的终点分析。如本文所用，术语基因组拷贝(GC)和载体基因组(vg)在剂量或剂量(例如，GC/kg和vg/kg)的上下文中是可互换的。Conventional methods for characterizing or quantifying rAAV are available to those skilled in the art. In order to calculate the empty and full particle content, the VP3 band volume of a selected sample (e.g., in the examples herein, a preparation purified by iodixanol gradient, wherein the number of GCs = the number of particles) is plotted relative to the attached GC particles. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test sample peak. The number of particles (pt) per 20 μL load is then multiplied by 50 to obtain particles (pt)/mL. Pt/mL is divided by GC/mL to obtain the ratio of particles to genome copies (pt/GC). Pt/mL-GC/mL obtains empty pt/mL. Empty pt/mL is divided by pt/mL, x100 to obtain the percentage of empty particles. Generally, methods for determining empty capsids and AAV vector particles using packaged genomes are known in the art. See, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method comprises subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins, e.g., a gradient gel containing 3 to 8% Tris-acetate in a buffer, then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. An anti-AAV capsid antibody is then used as a primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., Journal of Virology (2000) 74:9281-9293). Then use a second antibody, which is combined with the first antibody and contains a means for detecting the combination with the first antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound thereto, most preferably a goat anti-mouse IgG antibody covalently linked to horseradish peroxidase. The method for detecting the combination is used for semi-quantitative determination of the combination between the first antibody and the second antibody, preferably a detection method capable of detecting radioisotope emission, electromagnetic radiation or colorimetric changes, most preferably a chemiluminescent detection kit. For example, for SDS-PAGE, a sample can be taken out from the column fraction and heated in an SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and the capsid protein is resolved on a prefabricated gradient polyacrylamide gel (e.g., Novex). SilverXpress (Invitrogen, California) can be used according to the manufacturer's instructions or other suitable staining methods (i.e., SYPRO ruby or Coomassie staining) for silver staining. In one embodiment, the concentration of the AAV vector genome (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After nuclease inactivation, the sample is further diluted and amplified using a TaqMan^™ fluorescent probe specific for the DNA sequence between primers and primers. The number of cycles (threshold cycle, Ct) required for each sample to reach a specified fluorescence level is measured on an Applied Biosystems Prism 7700 sequence detection system. A standard curve is generated in a Q-PCR reaction using plasmid DNA containing the same sequence as that contained in the AAV vector. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by standardizing it to the Ct value of the plasmid standard curve. End-point analysis based on digital PCR can also be used. As used herein, the terms genome copy (GC) and vector genome (vg) are interchangeable in the context of dosage or dosage (e.g., GC/kg and vg/kg).

在一个方面，使用优化的q-PCR方法，其利用广谱丝氨酸蛋白酶，例如蛋白酶K(如可购自凯杰公司(Qiagen))。更具体地，优化的qPCR基因组滴度测定类似于标准测定，除了在DNase I消化后，将样品用蛋白酶K缓冲液稀释并用蛋白酶K处理，然后热灭活。合适地，用蛋白酶K缓冲液以等于样品大小的量稀释样品。蛋白酶K缓冲液可以浓缩至2倍或更高。通常，蛋白酶K处理为约0.2mg/mL，但可以在0.1mg/mL至约1mg/mL之间变化。处理步骤通常在约55℃下进行约15分钟，但可以在较低温度(例如，约37℃至约50℃)下进行较长时段(例如，约20分钟至约30分钟)，或在较高温度(例如，高达约60℃)下进行较短时段(例如，约5至10分钟)。类似地，热灭活通常在约95℃下持续约15分钟，但可以降低温度(例如，约70至约90℃)并延长时间(例如，约20分钟至约30分钟)。然后将样品稀释(例如，1000倍)并如标准测定中所述进行TaqMan分析。In one aspect, an optimized q-PCR method is used, which utilizes a broad spectrum of serine proteases, such as proteinase K (such as available from Qiagen). More specifically, the optimized qPCR genomic titer assay is similar to a standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, and then heat inactivated. Suitably, the sample is diluted with proteinase K buffer in an amount equal to the sample size. Proteinase K buffer can be concentrated to 2 times or higher. Typically, proteinase K is treated with about 0.2 mg/mL, but can vary between 0.1 mg/mL and about 1 mg/mL. The treatment step is usually performed at about 55°C for about 15 minutes, but can be performed at a lower temperature (e.g., about 37°C to about 50°C) for a longer period (e.g., about 20 minutes to about 30 minutes), or at a higher temperature (e.g., up to about 60°C) for a shorter period (e.g., about 5 to 10 minutes). Similarly, heat inactivation is typically performed at about 95°C for about 15 minutes, but the temperature can be lowered (e.g., about 70 to about 90°C) and the time can be extended (e.g., about 20 to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.

另外或替代地，可以使用液滴数字PCR(ddPCR)。例如，已经描述了通过ddPCR测定单链和自互补AAV载体基因组滴度的方法。参见，例如，M.Lock等人，《人类基因治疗方法》，《人类基因治疗方法》2014年4月；25(2)：115-25.doi：10.1089/hgtb.2013.131.电子版2014年2月14日。Additionally or alternatively, droplet digital PCR (ddPCR) can be used. For example, methods for determining the titer of single-stranded and self-complementary AAV vector genomes by ddPCR have been described. See, e.g., M. Lock et al., Human Gene Therapy Approaches, Human Gene Therapy Approaches Apr 2014;25(2):115-25. doi:10.1089/hgtb.2013.131. Epub 2014 Feb 14.

用于测定衣壳蛋白的vp1、vp2和vp3之间的比率的方法也是可用的。参见，例如，Vamseedhar Rayaprolu等人，《腺相关病毒衣壳稳定性和动力学的比较分析(ComparativeAnalysis of Adeno-Associated Virus Capsid Stability and Dynamics)》，《病毒学杂志》2013年12月；87(24)：13150-13160；Buller RM，Rose JA.1978.《KB细胞中腺病毒相关病毒诱导多肽的特征(Characterization of adenovirus-associated virus-inducedpolypeptides in KB cells)》《病毒学杂志》25：331-338；和Rose JA，Maizel JV，InmanJK，Shatkin AJ.1971.《腺病毒相关病毒的结构蛋白(Structural proteins ofadenovirus-associated viruses)》《病毒学杂志》8：766-770.Methods for determining the ratios between vp1, vp2, and vp3 of capsid proteins are also available. See, e.g., Vamseedhar Rayaprolu et al., Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics, Journal of Virology 2013 Dec;87(24):13150-13160; Buller RM, Rose JA. 1978. Characterization of adenovirus-associated virus-induced polypeptides in KB cells, Journal of Virology 25:331-338; and Rose JA, Maizel JV, Inman JK, Shatkin AJ. 1971. Structural proteins of adenovirus-associated viruses, Journal of Virology 8:766-770.

如本文所用，术语“治疗(treatment)”或“治疗(treating)”是指用于缓解法布里病的一种或多种症状、恢复hGLA的所需功能或改善疾病的生物标志物的目的的组合物和/或方法。在一些实施例中，术语“治疗(treatment)”或“治疗(treating)”被定义为涵盖出于本文指示的目的向受试者施用一种或多种本文所述的组合物。因此，“治疗”可以包括在给定受试者中减少法布里病的发作或进展、预防疾病、降低疾病症状的严重性、延缓其进展、消除疾病症状、延缓疾病的进展或增加治疗疗效中的一种或多种。As used herein, the term "treatment" or "treating" refers to compositions and/or methods for the purpose of alleviating one or more symptoms of Fabry disease, restoring the desired function of hGLA, or improving biomarkers of disease. In some embodiments, the term "treatment" or "treating" is defined to cover administering one or more compositions described herein to a subject for the purposes indicated herein. Thus, "treatment" may include reducing the onset or progression of Fabry disease, preventing the disease, reducing the severity of disease symptoms, delaying its progression, eliminating disease symptoms, delaying the progression of the disease, or increasing the efficacy of treatment in a given subject.

应当理解，本文所述rAAV中的组合物旨在应用于说明书中描述的其它组合物、方案、方面、实施例和方法。It should be understood that the compositions in rAAV described herein are intended to be applicable to other compositions, schemes, aspects, embodiments and methods described in the specification.

5.药物组合物或制剂5. Pharmaceutical compositions or preparations

在某些实施例中，本文提供了一种药物组合物，其在制剂缓冲液中包含本文所述的载体，如rAAV。在某些实施例中，药物组合物适于与功能性hGLA蛋白(ERT)(例如Fabrazyme)或伴侣疗法(例如Galafold(米加司他(migalastat))、爱美医疗公司(AmicusTherapeutics))共同施用。在一个实施例中，提供了一种药物组合物，其在制剂缓冲液中包含本文所述的rAAV。在某些实施例中，rAAV以约1x10⁹基因组拷贝(GC)/mL至约1x10¹⁴GC/mL配制。在另一实施例中，rAAV以约3x10⁹GC/mL至约3x10¹³GC/mL配制。在又一实施例中，rAAV以约1x10⁹GC/mL至约1x10¹³GC/mL配制。在一个实施例中，rAAV以至少约1x10¹¹GC/mL配制。In certain embodiments, a pharmaceutical composition is provided herein, which comprises a vector as described herein, such as rAAV, in a formulation buffer. In certain embodiments, the pharmaceutical composition is suitable for co-administration with a functional hGLA protein (ERT) (e.g., Fabrazyme) or a companion therapy (e.g., Galafold (migalastat), Amicus Therapeutics). In one embodiment, a pharmaceutical composition is provided, which comprises rAAV as described herein in a formulation buffer. In certain embodiments, rAAV is prepared at about 1x10⁹ genome copies (GC)/mL to about 1x10¹⁴ GC/mL. In another embodiment, rAAV is prepared at about 3x10⁹ GC/mL to about 3x10 13 GC/mL. In yet another embodiment, rAAV is prepared at about 1x10⁹ GC^{/mL to about 1x10 13 GC/mL. In one embodiment, rAAV is prepared at least about 1x10 11GC/} mL.

在某些实施例中，药物组合物包含在非病毒或病毒载体系统中包含hGLA编码序列的表达盒。这可以包括例如裸DNA、裸RNA、无机颗粒、脂质或脂质样颗粒、基于壳聚糖的制剂和本领域已知的和例如由Ramamoorth和Narvekar描述的其它制剂，如上所述)。这种非病毒载体系统可以包括例如质粒或非病毒遗传元件，或基于蛋白质的载体。In certain embodiments, the pharmaceutical composition comprises an expression cassette comprising an hGLA coding sequence in a non-viral or viral vector system. This may include, for example, naked DNA, naked RNA, inorganic particles, lipid or lipid-like particles, chitosan-based formulations, and other formulations known in the art and described, for example, by Ramamoorth and Narvekar, as described above). Such non-viral vector systems may include, for example, plasmids or non-viral genetic elements, or protein-based vectors.

在某些实施例中，药物组合物包含非复制型病毒载体。合适的病毒载体可以包括任何合适的递送载体，例如重组腺病毒、重组慢病毒、重组博卡病毒、重组腺相关病毒(AAV)或另一种重组细小病毒。在某些实施例中，病毒载体是用于将hGLA递送至有需要的患者的重组AAV。In certain embodiments, the pharmaceutical composition comprises a non-replicating viral vector. Suitable viral vectors may include any suitable delivery vector, such as a recombinant adenovirus, a recombinant lentivirus, a recombinant bocavirus, a recombinant adeno-associated virus (AAV), or another recombinant parvovirus. In certain embodiments, the viral vector is a recombinant AAV for delivering hGLA to a patient in need.

如本文所用，rAAV“原种”是指rAAV群体。尽管它们的衣壳蛋白由于脱酰胺作用而具有异质性，但是预计原种中的rAAV共有相同的载体基因组。原种可以包括具有衣壳的rAAV，这些衣壳具有例如所选AAV衣壳蛋白和所选生产系统的异质脱酰胺模式特征。原种可以由单个生产系统生产或由生产系统的多个运行汇集。可以选择各种生产系统，包括但不限于本文所述的那些。As used herein, rAAV "stock" refers to a rAAV population. Although their capsid proteins are heterogeneous due to deamidation, it is expected that the rAAVs in the stock have the same vector genome. The stock can include rAAVs with capsids that have, for example, heterogeneous deamidation patterns characteristic of a selected AAV capsid protein and a selected production system. The stock can be produced by a single production system or collected by multiple runs of a production system. Various production systems can be selected, including but not limited to those described herein.

在一个实施例中，药物组合物包含载体和制剂缓冲液，载体包括包含hGLA编码序列的表达盒，制剂缓冲液适于通过脑室内(ICV)、鞘内(IT)、脑池内或静脉内(IV)注射递送。在一个实施例中，包含hGLA编码序列的表达盒包装在重组AAV中。In one embodiment, the pharmaceutical composition comprises a vector including an expression cassette comprising an hGLA coding sequence and a formulation buffer suitable for delivery by intracerebroventricular (ICV), intrathecal (IT), intracisternal or intravenous (IV) injection. In one embodiment, the expression cassette comprising the hGLA coding sequence is packaged in a recombinant AAV.

在一个实施例中，药物组合物包含功能性hGLA多肽或其功能性片段，用于作为酶替代疗法(ERT)递送至受试者。这种药物组合物通常静脉内施用，然而在一些情况下皮内、肌内或口服施用也是可能的。该组合物可用于预防性治疗患有法布里病或处于法布里病危险中的个体。对于治疗应用，将药物组合物以足以降低累积的代谢物的浓度和/或预防或阻止代谢物的进一步累积的量施用于患有确定疾病的患者。对于处于溶酶体酶缺乏症风险中的个体，以足以预防或抑制代谢物累积的量预防性地施用药物组合物。包含本文所述的hGLA蛋白的药物组合物以治疗有效量施用。通常，治疗有效量可以根据受试者的医学病状的严重程度以及受试者的年龄、总体状况和性别而变化。剂量可以由医师确定并且可以根据需要进行调整以适合所观察到的治疗效果。在一个方面，本文提供了配制成含有单位剂量的hGLA蛋白或其功能片段的用于ERT的药物组合物。In one embodiment, the pharmaceutical composition comprises a functional hGLA polypeptide or a functional fragment thereof for delivery to a subject as an enzyme replacement therapy (ERT). This pharmaceutical composition is usually administered intravenously, but in some cases intradermal, intramuscular or oral administration is also possible. The composition can be used for the prophylactic treatment of individuals suffering from Fabry disease or at risk of Fabry disease. For therapeutic applications, the pharmaceutical composition is administered to a patient with a determined disease in an amount sufficient to reduce the concentration of the accumulated metabolites and/or prevent or prevent the further accumulation of metabolites. For individuals at risk of lysosomal enzyme deficiency, the pharmaceutical composition is prophylactically administered in an amount sufficient to prevent or inhibit the accumulation of metabolites. The pharmaceutical composition comprising the hGLA protein described herein is administered in a therapeutically effective amount. Generally, the therapeutically effective amount can vary according to the severity of the subject's medical condition and the subject's age, overall condition and gender. The dosage can be determined by a physician and can be adjusted as needed to suit the observed therapeutic effect. In one aspect, a pharmaceutical composition for ERT formulated to contain a unit dose of hGLA protein or a functional fragment thereof is provided herein.

在某些实施例中，制剂进一步包含溶解在水性悬浮液中的表面活性剂、防腐剂、赋形剂和/或缓冲液。在一个实施例中，缓冲液是PBS。在另一实施例中，缓冲液是人工脑脊液(aCSF)，例如艾略特制剂缓冲液(Eliott′s formulation buffer)；或哈佛仪器灌注流体(Harvard apparatus perfusion fluid)(具有最终离子浓度(以mM计)的人工CSF：Na 150；K 3.0；Ca 1.4；Mg 0.8；P 1.0；C1155)。各种合适的溶液是已知的，包括那些包括以下一种或多种的溶液：缓冲盐水、表面活性剂和调节至相当于约100mM氯化钠(NaCl)至约250mM氯化钠的离子强度的生理上相容的盐或盐的混合物，或调节至相当于离子浓度的生理上相容的盐。In certain embodiments, the formulation further comprises a surfactant, a preservative, an excipient, and/or a buffer dissolved in an aqueous suspension. In one embodiment, the buffer is PBS. In another embodiment, the buffer is an artificial cerebrospinal fluid (aCSF), such as Eliott's formulation buffer; or Harvard apparatus perfusion fluid (artificial CSF with a final ionic concentration (in mM): Na 150; K 3.0; Ca 1.4; Mg 0.8; P 1.0; C1155). Various suitable solutions are known, including those comprising one or more of the following: a buffered saline, a surfactant, and a physiologically compatible salt or mixture of salts adjusted to an ionic strength equivalent to about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or a physiologically compatible salt adjusted to an ionic concentration equivalent to.

合适地，将制剂调节至生理上可接受的pH，例如在pH 6至8，或pH 6.5至7.5，pH7.0至7.7，或pH 7.2至7.8的范围内。由于脑脊液的pH为约7.28至约7.32，对于鞘内递送，在此范围内的pH可能是期望的；而对于静脉内递送，pH为6.8至约7.2可能是期望的。然而，在最宽范围和这些子范围内的其它pH可以选择用于其它递送途径。Suitably, the formulation is adjusted to a physiologically acceptable pH, for example in the range of pH 6 to 8, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Since the pH of cerebrospinal fluid is about 7.28 to about 7.32, a pH in this range may be desirable for intrathecal delivery, while a pH of 6.8 to about 7.2 may be desirable for intravenous delivery. However, other pHs within the broadest range and these subranges may be selected for other delivery routes.

合适的表面活性剂或表面活性剂的组合可以选自无毒的非离子表面活性剂。在一个实施例中，选择终止于伯羟基的双官能嵌段共聚物表面活性剂，例如F68[BASF]，也称为泊洛沙姆(Poloxamer)188，其具有中性pH，具有8400的平均分子量。可以选择其它表面活性剂和其它泊洛沙姆，即由侧翼为两个聚氧乙烯(聚(环氧乙烷))的亲水链的聚氧丙烯(聚(环氧丙烷))的中心疏水链、SOLUTOL HS 15(聚乙二醇-15羟基硬脂酸酯)、LABRASOL(聚氧辛酸甘油酯)、聚氧10油基醚、TWEEN(聚氧乙烯山梨醇脂肪酸酯)、乙醇和聚乙二醇组成的非离子三嵌段共聚物。在一个实施例中，制剂含有泊洛沙姆。这些共聚物通常用字母“P”(对于泊洛沙姆)后跟三个数字命名：前两个数字×100得到聚氧丙烯核的近似分子量，最后一个数字×10得到聚氧乙烯含量的百分比。在一个实施例中，选择泊洛沙姆188。表面活性剂可以以悬浮液的至多约0.0005％至约0.001％的量存在。Suitable surfactants or combinations of surfactants may be selected from non-toxic nonionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated in a primary hydroxyl group is selected, such as F68 [BASF], also known as Poloxamer 188, has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., a nonionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxycaprylyl glyceride), polyoxyl 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are usually named with the letter "P" (for poloxamer) followed by three numbers: the first two numbers x 100 give the approximate molecular weight of the polyoxypropylene core, and the last number x 10 gives the percentage of the polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

在一个实例中，制剂可以含有例如缓冲盐水溶液，该缓冲盐水溶液在水中包含氯化钠、碳酸氢钠、右旋糖、硫酸镁(例如，硫酸镁·7H2O)、氯化钾、氯化钙(例如，氯化钙·2H2O)、磷酸氢二钠及其混合物中的一种或多种。合适地，对于鞘内递送，渗透压在与脑脊液相容的范围内(例如，约275至约290)；参见例如，emedicine.medscape.com/article/2093316-overview。任选地，对于鞘内递送，市售稀释剂可用作悬浮剂，或与另一悬浮剂和其它任选赋形剂组合使用。参见，例如，Elliotts溶液[Lukare医疗公司]。In one example, the formulation can contain, for example, a buffered saline solution comprising one or more of sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate·7H2O), potassium chloride, calcium chloride (e.g., calcium chloride·2H2O), disodium hydrogen phosphate, and mixtures thereof in water. Suitably, for intrathecal delivery, the osmotic pressure is within a range compatible with cerebrospinal fluid (e.g., about 275 to about 290); see, e.g., emedicine.medscape.com/article/2093316-overview. Optionally, for intrathecal delivery, a commercially available diluent may be used as a suspending agent, or used in combination with another suspending agent and other optional excipients. See, e.g., Elliotts Solution [Lukare Medical Corporation].

在某些实施例中，制剂可以含有一种或多种渗透增强剂。合适的渗透增强剂的实例可以包括例如甘露醇、甘胆酸钠、牛磺胆酸钠、脱氧胆酸钠、水杨酸钠、辛酸钠、癸酸钠、十二烷基硫酸钠、聚氧乙烯-9-月桂基醚或EDTA。In certain embodiments, the formulation may contain one or more penetration enhancers. Examples of suitable penetration enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium octanoate, sodium decanoate, sodium dodecyl sulfate, polyoxyethylene-9-lauryl ether or EDTA.

在一个实施例中，提供了冷冻形式的冷冻组合物，其含有本文所述的在缓冲溶液中的rAAV。任选地，一种或多种表面活性剂(例如，普朗尼克(Pluronic)F68)、稳定剂或防腐剂存在于该组合物中。合适地，为了使用，将组合物解冻并用合适的稀释剂(例如，无菌盐水或缓冲盐水)滴定至所需剂量。In one embodiment, a frozen composition is provided in frozen form, which contains rAAV as described herein in a buffered solution. Optionally, one or more surfactants (e.g., Pluronic F68), stabilizers or preservatives are present in the composition. Suitably, for use, the composition is thawed and titrated to the desired dose with a suitable diluent (e.g., sterile saline or buffered saline).

在某些实施例中，本文提供了一种药物组合物，其包含本文所述的载体(如rAAV)和药学上可接受的载体。如本文所用，“载体”包括任何和所有溶剂、分散介质、媒介物、包衣、稀释剂、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂、缓冲液、载体溶液、悬浮液、胶体等。此类介质和药剂用于药物活性物质的用途是本领域熟知的。辅助活性成分也可以掺入组合物中。递送媒介物(如脂质体、纳米胶囊、微粒、微球、脂质颗粒、囊泡等)可用于将本发明的组合物引入合适的宿主细胞。特别地，rAAV载体可以配制成包封在脂质颗粒、脂质体、囊泡、纳米球或纳米颗粒等中的递送形式。在一个实施例中，治疗有效量的所述载体包括在药物组合物中。载体的选择不是对本发明的限制。其它常规的药学上可接受的载体，如防腐剂或化学稳定剂。合适的示例性防腐剂包括氯丁醇、山梨酸钾、山梨酸、二氧化硫、没食子酸丙酯、对羟基苯甲酸酯、乙基香草醛、甘油、苯酚和对氯苯酚。合适的化学稳定剂包括明胶和白蛋白。In certain embodiments, a pharmaceutical composition is provided herein, comprising a vector (such as rAAV) described herein and a pharmaceutically acceptable carrier. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic agents and absorption delay agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Auxiliary active ingredients can also be incorporated into the composition. Delivery vehicles (such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc.) can be used to introduce the composition of the present invention into suitable host cells. In particular, the rAAV vector can be formulated into a delivery form encapsulated in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc. In one embodiment, a therapeutically effective amount of the carrier is included in a pharmaceutical composition. The choice of carrier is not a limitation of the present invention. Other conventional pharmaceutically acceptable carriers, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

短语“药学上可接受的”是指当施用于宿主时不产生过敏或类似不良反应的分子实体和组合物。The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

如本文所用，术语“剂量”或“量”可以指在治疗过程中递送至受试者的总剂量或量，或在单一单位(或多个单位或分开的剂量)施用中递送的剂量或量。As used herein, the term "dose" or "amount" may refer to the total dose or amount delivered to a subject during the course of treatment, or the dose or amount delivered in a single unit (or multiple units or divided doses) administration.

而且，复制缺陷型病毒组合物可以配制成含有复制缺陷型病毒的剂量单位，复制缺陷型病毒的量在约1.0x10⁹GC至约1.0x10¹⁶Gc的范围内(以治疗平均体重为70kg的受试者)，包括该范围内的所有整数或分数，对于人类患者优选地1.0x10¹²GC至1.0x10¹⁴GC。在一个实施例中，将组合物配制成每剂量含有至少1x10⁹、2x10⁹、3x10⁹、4x10⁹、5x10⁹、6x10⁹、7x10⁹、8x10⁹或9x10⁹GC，包括该范围内的所有整数或分数量。在另一实施例中，将组合物配制成每剂量含有至少1x10¹⁰、2x10¹⁰、3x10¹⁰、4x10¹⁰、5x10¹⁰、6x10¹⁰、7x10¹⁰、8x10¹⁰或9x10¹⁰GC，包括该范围内的所有整数或分数量。在另一实施例中，将组合物配制成每剂量含有至少1x10¹¹、2x10¹¹、3x10¹¹、4x10¹¹、5x10¹¹、6x10¹¹、7x10¹¹、8x10¹¹或9x10¹¹GC，包括该范围内的所有整数或分数量。在另一实施例中，将组合物配制成每剂量含有至少1x10¹²、2x10¹²、3x10¹²、4x10¹²、5x10¹²、6x10¹²、7x10¹²、8x10¹²或9x10¹²GC，包括该范围内的所有整数或分数量。在另一实施例中，将组合物配制成每剂量含有至少1x10¹³、2x10¹³、3x10¹³、4x10¹³、5x10¹³、6x10¹³、7x10¹³、8x10¹³或9x10¹³GC，包括该范围内的所有整数或分数量。在另一实施例中，将组合物配制成每剂量含有至少1x10¹⁴、2x10¹⁴、3x10¹⁴、4x10¹⁴、5x10¹⁴、6x10¹⁴、7x10¹⁴、8x10¹⁴或9x10¹⁴GC，包括该范围内的所有整数或分数量。在另一实施例中，将组合物配制成每剂量含有至少1x10¹⁵、2x10¹⁵、3x10¹⁵、4x10¹⁵、5x10¹⁵、6x10¹⁵、7x10¹⁵、8x10¹⁵或9x10¹⁵GC，包括该范围内的所有整数或分数量。在一个实施例中，对于人类应用，剂量可以为每剂量1x10¹⁰至约1x10¹²GC，包括该范围内的所有整数或分数量。Moreover, the replication-defective virus composition can be formulated as a dosage unit containing the replication-defective virus, the amount of the replication-defective virus is in the range of about 1.0x10⁹ GC to about 1.0x10¹⁶ Gc (to treat a subject with an average body weight of 70 kg), including all integers or fractions within this range, preferably 1.0x10¹² GC to 1.0x10¹⁴ GC for human patients. In one embodiment, the composition is formulated to contain at least 1x10⁹ , 2x10⁹ , 3x10⁹ , 4x10⁹ , 5x10⁹ , 6x10⁹ , 7x10⁹ , 8x10⁹ or 9x10⁹ GC per dose, including all integers or fractions within this range. In another embodiment, the composition is formulated to contain at least^1x1010 ,^2x1010 ,^3x1010 , 4x1010,^5x1010 ,^6x1010 ,^7x1010 ,^8x1010 , or^9x1010 GC per dose, including all integers or fractional amounts within that range. In another embodiment, the composition is formulated to contain at least^1x1011 ,^2x1011 ,^3x1011 ,^4x1011 ,^{5x1011,6x1011} ,^7x1011 ,^8x1011 , or^9x1011 GC per dose, including all integers or fractional amounts within that range. In another embodiment, the composition is formulated to contain at least^1x1012 ,^2x1012 ,^3x1012 , 4x1012,^5x1012 ,^6x1012 ,^7x1012 ,^8x1012 , or^9x1012 GC per dose, including all integer or fractional amounts within that range. In another embodiment, the composition is formulated to contain at least^1x1013 ,^2x1013 ,^3x1013 ,^4x1013 ,^{5x1013,6x1013} ,^7x1013 ,^8x1013 , or^9x1013 GC per dose, including all integer or fractional amounts within that range. In another embodiment, the composition is formulated to contain at least 1x10¹⁴ , 2x10¹⁴ , 3x10¹⁴ , 4x10¹⁴ , 5x10¹⁴ , 6x10¹⁴ , 7x10¹⁴ , 8x10¹⁴ , or 9x10¹⁴ GC per dose, including all integers or fractional amounts within this range. In another embodiment, the composition is formulated to contain at least 1x10¹⁵ , 2x10¹⁵ , 3x10¹⁵ , 4x10¹⁵ , 5x10¹⁵ , 6x10¹⁵ , 7x10¹⁵ , 8x10¹⁵ , or 9x10¹⁵ GC per dose, including all integers or fractional amounts within this range. In one embodiment, for human use, the dosage can be 1x10¹⁰ to about 1x10¹² GC per dose, including all integers or fractional amounts within this range.

在某些实施例中，提供了一种药物组合物，其在制剂缓冲液中包含本文所述的rAAV。在一个实施例中，rAAV以约1x10⁹基因组拷贝(GC)/mL至约1x10¹⁴GC/mL配制。在另一实施例中，rAAV以约3x10⁹GC/mL至约3x10¹³GC/mL配制。在又一实施例中，rAAV以约1x10⁹GC/mL至约1x10¹³GC/mL配制。在一个实施例中，rAAV以至少约1x10¹¹GC/mL配制。在一个实施例中，包含本文所述的rAAV的药物组合物可以以约1x10⁹GC/克脑质量至约1x10¹⁴GC/克脑质量的剂量施用。In certain embodiments, a pharmaceutical composition is provided, which comprises rAAV described herein in a formulation buffer. In one embodiment, rAAV is formulated at about 1x10⁹ genome copies (GC)/mL to about 1x10¹⁴ GC/mL. In another embodiment, rAAV is formulated at about 3x10⁹ GC/mL to about 3x10¹³ GC/mL. In yet another embodiment, rAAV is formulated at about 1x10⁹ GC/mL to about 1x10^{13 GC/mL. In one embodiment, rAAV is formulated at at least about 1x10 11GC} /mL. In one embodiment, a pharmaceutical composition comprising rAAV described herein can be administered at a dose of about 1x10⁹ GC/gram of brain mass to about 1x10¹⁴ GC/gram of brain mass.

在某些实施例中，组合物可以配制在合适的水性悬浮介质(例如，缓冲盐水)中用于通过任何合适的途径递送。本文提供的组合物可用于全身递送高剂量的病毒载体。对于rAAV，高剂量可以是至少1x10¹³GC或至少1x10¹⁴GC。然而，为了提高安全性，本文提供的miRNA序列可以包含在以其它较低剂量递送的表达盒和/或载体基因组中。In certain embodiments, the composition can be formulated in a suitable aqueous suspension medium (e.g., buffered saline) for delivery by any suitable route. The compositions provided herein can be used for systemic delivery of high-dose viral vectors. For rAAV, the high dose can be at least 1x10¹³ GC or at least 1x10¹⁴ GC. However, to improve safety, the miRNA sequences provided herein can be included in expression cassettes and/or vector genomes delivered at other lower doses.

本文所述的水性悬浮液或药物组合物被设计用于通过任何合适的途径或不同途径的组合递送至有需要的受试者。在一个实施例中，药物组合物被配制用于通过脑室内(ICV)、鞘内(IT)或脑池内注射递送。在一个实施例中，本文所述的组合物被设计用于通过静脉内(IV)注射递送至有需要的受试者。替代地，可以选择其它施用途径(例如，口服、吸入、鼻内、气管内、动脉内、眼内、肌内和其它肠胃外途径)。在某些实施例中，组合物基本上同时通过两种不同的途径递送。Aqueous suspensions or pharmaceutical compositions described herein are designed to be delivered to a subject in need by any suitable route or combination of different routes. In one embodiment, the pharmaceutical composition is formulated for delivery by intracerebroventricular (ICV), intrathecal (IT) or intracisternal injection. In one embodiment, the compositions described herein are designed to be delivered to a subject in need by intravenous (IV) injection. Alternatively, other routes of administration (e.g., oral, inhaled, intranasal, intratracheal, intraarterial, intraocular, intramuscular and other parenteral routes) can be selected. In certain embodiments, the composition is delivered by two different routes substantially simultaneously.

如本文所用，术语“鞘内递送”或“鞘内施用”是指药物经由注射到椎管中，更具体地注射到蛛网膜下腔中使得其到达脑脊液(CSF)的施用途径。鞘内递送可以包括腰椎穿刺、心室内、枕下/脑池内和/或C1-2穿刺。例如，可以通过腰椎穿刺引入材料以扩散到整个蛛网膜下腔。在另一实例中，注射可以进入小脑延髓池。脑池内递送可以增加载体扩散和/或减少由施用引起的毒性和炎症。参见，例如，Christian Hinderer等人，《AAV9递送至小脑延髓池后在食蟹猴中枢神经系统的广泛基因转移(Widespread gene transfer in thecentral nervous system of cynomolgus macaques following delivery of AAV9 intothe cisterna magna)》，《分子治疗方法与临床开发(Mol Ther Methods Clin Dev.)》2014；1：14051.于2014年12月10日在线发表。doi：10.1038/mtm.2014.51.As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to a route of administration in which a drug is injected into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery can include lumbar puncture, intraventricular, suboccipital/intracisternal and/or C1-2 puncture. For example, the material can be introduced via a lumbar puncture to diffuse throughout the subarachnoid space. In another example, the injection can enter the cisterna magna. Intracisternal delivery can increase vector diffusion and/or reduce toxicity and inflammation caused by administration. See, e.g., Christian Hinderer et al., "Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna," Mol Ther Methods Clin Dev. 2014; 1: 14051. Published online Dec. 10, 2014. doi: 10.1038/mtm.2014.51.

如本文所用，术语“脑池内递送”或“脑池内施用”是指将药物直接施用于脑室的脑脊液中或小脑延髓池内的施用途径，更具体地通过枕下穿刺或通过直接注射到小脑延髓池中或通过永久定位的管。As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to a route of administration in which the drug is administered directly into the cerebrospinal fluid of the ventricles of the brain or into the cisterna magna, more specifically via a suboccipital puncture or by injection directly into the cisterna magna or via a permanently positioned cannula.

应当理解，本文所述药物组合物中的组合物旨在应用于说明书中描述的其它组合物、方案、方面、实施例和方法。It should be understood that the compositions within the pharmaceutical compositions described herein are intended to be applicable to other compositions, regimens, aspects, embodiments and methods described in the specification.

6.治疗方法6. Treatment Methods

本文提供了用于法布里病的方法，其包含递送治疗有效量的包括本文提供的hGLA编码序列的核酸序列或表达盒。具体地，该方法包括通过向有需要的患者递送治疗有效量的rAAV.hGLA或包括本文所述的hGLA多肽的组合物来预防、治疗和/或缓解法布里病的症状。在某些实施例中，将包含本文所述的表达盒的组合物施用于有需要的受试者。在某些实施例中，表达盒通过rAAV递送。Provided herein is a method for Fabry disease, comprising delivering a therapeutically effective amount of a nucleic acid sequence or expression cassette comprising an hGLA coding sequence provided herein. Specifically, the method comprises preventing, treating and/or alleviating the symptoms of Fabry disease by delivering a therapeutically effective amount of rAAV.hGLA or a composition comprising an hGLA polypeptide as described herein to a patient in need. In certain embodiments, a composition comprising an expression cassette as described herein is administered to a subject in need. In certain embodiments, the expression cassette is delivered by rAAV.

如本文所用，“治疗有效量”是指递送足以缓解或治疗法布里病的一种或多种症状的量的hGLA的组合物的量。“治疗”可以包括预防法布里病症状的恶化和可能逆转其一种或多种症状。人类患者的“治疗有效量”可以基于动物模型来预测。参见，C.Hinderer等人，《分子疗法》(2014)；2212，2018-2027；A.Bradbury等人，《人类基因疗法临床发展(Human GeneTherapy Clinical Development)》2015年3月，26(1)：27-37，其通过引用并入本文。As used herein, "therapeutically effective amount" refers to the amount of a composition that delivers an amount of hGLA sufficient to alleviate or treat one or more symptoms of Fabry disease. "Treatment" can include preventing the worsening of symptoms of Fabry disease and possibly reversing one or more symptoms thereof. A "therapeutically effective amount" for human patients can be predicted based on animal models. See, C. Hinderer et al., Molecular Therapy (2014); 2212, 2018-2027; A. Bradbury et al., Human Gene Therapy Clinical Development, March 2015, 26(1): 27-37, which are incorporated herein by reference.

在某些实施例中，治疗包括预防、治疗和/或缓解法布里病的一种或多种症状，包括例如肾病、心肌病、疼痛、疲劳、中风、听力损失、胃肠病症。In certain embodiments, treatment includes preventing, curing and/or ameliorating one or more symptoms of Fabry disease including, for example, renal disease, cardiomyopathy, pain, fatigue, stroke, hearing loss, gastrointestinal disorders.

在某些实施例中，治疗包括将本文所述的表达盒、核酸、载体(例如rAAV)或多肽递送至微脉管系统、肾细胞、心脏/心脏细胞、外周神经和中枢神经系统的细胞中的一种或多种。在某些实施例中，治疗导致心肌细胞、足细胞、血管内皮细胞和背根神经节中的一种或多种中的α-GalA底物减少。在某些实施例中，治疗导致肾脏中α-GalA底物减少。在某些实施例中，治疗导致肾小管中α-GalA底物减少。In certain embodiments, treatment includes delivery of expression cassettes, nucleic acids, vectors (e.g., rAAV) or polypeptides as described herein to one or more of cells of the microvasculature, kidney cells, heart/cardiac cells, peripheral nerves, and central nervous system. In certain embodiments, treatment results in a reduction in α-GalA substrates in one or more of cardiomyocytes, podocytes, vascular endothelial cells, and dorsal root ganglia. In certain embodiments, treatment results in a reduction in α-GalA substrates in the kidney. In certain embodiments, treatment results in a reduction in α-GalA substrates in the renal tubules.

在某些实施例中，治疗包括通过基于rAAV的基因疗法替换或补充患者的缺陷型α-半乳糖苷酶A。如本文所述的rAAV载体所表达的，在CSF、血清、神经元或其它组织或流体中检测到的正常水平的至少约2％的表达水平可以提供治疗效果。然而，可以实现更高的表达水平。这种表达水平可以是正常功能性人GLA水平的2％至约100％。在某些实施例中，可以在血清或另一种生物流体或组织中检测到高于正常的表达水平。In certain embodiments, treatment includes replacing or supplementing the patient's defective α-galactosidase A by rAAV-based gene therapy. As expressed by the rAAV vector described herein, an expression level of at least about 2% of the normal level detected in CSF, serum, neurons, or other tissues or fluids can provide a therapeutic effect. However, higher expression levels can be achieved. This expression level can be 2% to about 100% of the normal functional human GLA level. In certain embodiments, higher than normal expression levels can be detected in serum or another biological fluid or tissue.

如本文所用，术语“NAb滴度”是产生多少中和抗体(例如，抗AAV Nab)的量度，该中和抗体中和其靶向表位(例如，AAV)的生理效应。抗AAV NAb滴度可以如Calcedo，R.等人，《腺相关病毒中和抗体的全球流行病学(Worldwide Epidemiology of NeutralizingAntibodies to Adeno-Associated Viruses)》《传染病杂志(Journal of InfectiousDiseases)》，2009。199(3)：第381至390页中所述进行测量，其通过引用并入本文。As used herein, the term "NAb titer" is a measure of how many neutralizing antibodies (e.g., anti-AAV NAbs) are produced that neutralize the physiological effects of their targeted epitopes (e.g., AAV). Anti-AAV NAb titers can be measured as described in Calcedo, R. et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses, Journal of Infectious Diseases, 2009. 199(3): pp. 381-390, which is incorporated herein by reference.

在某些实施例中，本文提供的组合物可用于将所需功能的hGLA产物递送至患者，同时抑制背根神经节神经元中的基因和/或基因产物的表达。在某些实施例中，该方法包括向患者递送包含表达盒的组合物，该表达盒包含hGLA编码序列和miRNA靶序列。在某些实施例中，该方法包含递送包括miR-183靶序列的表达盒或载体基因组以抑制DRG中的转基因表达水平。在某些实施例中，该方法包含递送用于抑制DRG中转基因表达的表达盒，其中该表达盒包括至少两个miR183靶序列、至少三个miR183靶序列、至少四个miR183靶序列、至少五个miR183靶序列、至少六个miR183靶序列、至少七个miR183靶序列或至少八个miR183靶序列。在某些实施例中，该方法包含递送用于抑制DRG中转基因表达的表达盒，其中该表达盒包括至少两个miR182靶序列、至少三个miR182靶序列、至少四个miR182靶序列、至少五个miR182靶序列、至少六个miR182靶序列、至少七个miR182靶序列或至少八个miR182靶序列。在某些实施例中，该表达盒包括一个或多个miR182靶序列和一个或多个miR183靶序列。In certain embodiments, the compositions provided herein can be used for delivering the hGLA product of the desired function to the patient, while suppressing the expression of the gene and/or gene product in the dorsal root ganglion neurons. In certain embodiments, the method includes delivering a composition comprising an expression cassette to the patient, the expression cassette comprising an hGLA coding sequence and a miRNA target sequence. In certain embodiments, the method includes delivering an expression cassette or a vector genome comprising a miR-183 target sequence to suppress the transgenic expression level in the DRG. In certain embodiments, the method includes delivering an expression cassette for suppressing transgenic expression in the DRG, wherein the expression cassette comprises at least two miR183 target sequences, at least three miR183 target sequences, at least four miR183 target sequences, at least five miR183 target sequences, at least six miR183 target sequences, at least seven miR183 target sequences or at least eight miR183 target sequences. In certain embodiments, the method comprises delivering an expression cassette for inhibiting transgenic expression in DRG, wherein the expression cassette comprises at least two miR182 target sequences, at least three miR182 target sequences, at least four miR182 target sequences, at least five miR182 target sequences, at least six miR182 target sequences, at least seven miR182 target sequences or at least eight miR182 target sequences. In certain embodiments, the expression cassette comprises one or more miR182 target sequences and one or more miR183 target sequences.

所提供的组合物的合适递送体积及其浓度可以由本领域技术人员确定。例如，可以选择约1μL至150mL的体积，对于成人选择更高的体积。通常，对于新生婴儿，合适的体积为约0.5mL至约10mL，对于年龄较大的婴儿，可以选择约0.5mL至约15mL。对于幼儿，可选择约0.5mL至约20mL的体积。对于儿童，可以选择高达约30mL的体积。对于青春期前和青少年，可以选择高达约50mL的体积。在其它实施例中，患者可以接受选定体积为约5mL至约15mL，或约7.5mL至约10mL的鞘内施用。可以确定其它合适的体积和剂量。调节剂量以平衡治疗益处与任何副作用，并且这种剂量可以根据使用重组载体的治疗应用而变化。The suitable delivery volume and concentration of the provided composition can be determined by those skilled in the art. For example, a volume of about 1 μ L to 150 mL can be selected, and a higher volume can be selected for adults. Typically, for newborn infants, a suitable volume is about 0.5 mL to about 10 mL, and for older infants, about 0.5 mL to about 15 mL can be selected. For young children, a volume of about 0.5 mL to about 20 mL can be selected. For children, a volume of up to about 30 mL can be selected. For prepubertal and adolescents, a volume of up to about 50 mL can be selected. In other embodiments, the patient can receive a selected volume of about 5 mL to about 15 mL, or about 7.5 mL to about 10 mL of intrathecal administration. Other suitable volumes and dosages can be determined. The dosage is adjusted to balance the therapeutic benefit with any side effects, and this dosage can vary according to the therapeutic application using the recombinant vector.

在某些实施例中，包含本文所述的rAAV的组合物可以以约1x10⁹GC/克脑质量至约1x10¹⁴GC/克脑质量的剂量施用。在某些实施例中，rAAV以约1x10⁹GC/kg体重至约1x10¹³GC/kg体重的剂量全身共同施用。In certain embodiments, a composition comprising a rAAV described herein can be administered at a dose of about^1x109 GC/gram brain mass to about^1x1014 GC/gram brain mass. In certain embodiments, rAAV is co-administered systemically at a dose of about^1x109 GC/kg body weight to about^1x1013 GC/kg body weight.

在某些实施例中，表达盒在载体基因组中的递送量为约1x10⁹GC/克脑质量至约1x10¹³基因组拷贝(GC)/克(g)脑质量，包括该范围和端点内的所有整数或分数量。在另一实施例中，剂量为1x10¹⁰GC/克脑质量至约1x10¹³GC/克脑质量。在具体的实施例中，施用于患者的载体的剂量为至少约1.0x10⁹GC/g、约1.5x10⁹GC/g、约2.0x10⁹GC/g、约2.5x10⁹GC/g、约3.0x10⁹GC/g、约3.5x10⁹GC/g、约4.0x10⁹GC/g、约4.5x10⁹GC/g、约5.0x10⁹GC/g、约5.5x10⁹GC/g、约6.0x10⁹GC/g、约6.5x10⁹GC/g、约7.0x10⁹GC/g、约7.5x10⁹GC/g、约8.0x10⁹GC/g、约8.5x10⁹GC/g、约9.0x10⁹GC/g、约9.5x10⁹GC/g、约1.0x10¹⁰GC/g、约1.5x10¹⁰GC/g、约2.0x10¹⁰GC/g、约2.5x10¹⁰GC/g、约3.0x10¹⁰GC/g、约3.5x10¹⁰GC/g、约4.0x10¹⁰GC/g、约4.5x10¹⁰GC/g、约5.0x10¹⁰GC/g、约5.5x10¹⁰GC/g、约6.0x10¹⁰GC/g、约6.5x10¹⁰GC/g、约7.0x10¹⁰GC/g、约7.5x10¹⁰GC/g、约8.0x10¹⁰GC/g、约8.5x10¹⁰GC/g、约9.0x10¹⁰GC/g、约9.5x10¹⁰GC/g、约1.0x10¹¹GC/g、约1.5x10¹¹GC/g、约2.0x10¹¹GC/g、约2.5x10¹¹GC/g、约3.0x10¹¹GC/g、约3.5x10¹¹GC/g、约4.0x10¹¹GC/g、约4.5x10¹¹GC/g、约5.0x10¹¹GC/g、约5.5x10¹¹GC/g、约6.0x10¹¹GC/g、约6.5x10¹¹GC/g、约7.0x10¹¹GC/g、约7.5x10¹¹GC/g、约8.0x10¹¹GC/g、约8.5x10¹¹GC/g、约9.0x10¹¹GC/g、约9.5x10¹¹GC/g、约1.0x10¹²GC/g、约1.5x10¹²GC/g、约2.0x10¹²GC/g、约2.5x10¹²GC/g、约3.0x10¹²GC/g、约3.5x10¹²GC/g、约4.0x10¹²GC/g、约4.5x10¹²GC/g、约5.0x10¹²GC/g、约5.5x10¹²GC/g、约6.0x10¹²GC/g、约6.5x10¹²GC/g、约7.0x10¹²GC/g、约7.5x10¹²GC/g、约8.0x10¹²GC/g、约8.5x10¹²GC/g、约9.0x10¹²GC/g、约9.5x10¹²GC/g、约1.0x10¹³GC/g、约1.5x10¹³GC/g、约2.0x10¹³GC/g、约2.5x10¹³GC/g、约3.0x10¹³GC/g、约3.5x10¹³GC/g、约4.0x10¹³GC/g、约4.5x10¹³GC/g、约5.0x10¹³GC/g、约5.5x10¹³GC/g、约6.0x10¹³GC/g、约6.5x10¹³GC/g、约7.0x10¹³GC/g、约7.5x10¹³GC/g、约8.0x10¹³GC/g、约8.5x10¹³GC/g、约9.0x10¹³GC/g、约9.5x10¹³GC/g或约1.0x10¹⁴GC/g脑质量。In certain embodiments, the expression cassette is delivered in the vector genome in an amount of about 1x10⁹ GC/gram brain mass to about 1x10¹³ genome copies (GC)/gram (g) brain mass, including all integer or fractional amounts within this range and endpoints. In another embodiment, the dosage is 1x10¹⁰ GC/gram brain mass to about 1x10¹³ GC/gram brain mass. In specific embodiments, the dose of the vector administered to a patient is at least about 1.0 x 10⁹ GC/g, about 1.5 x 10⁹ GC/g, about 2.0 x 10⁹ GC/g, about 2.5 x 10⁹ GC/g, about 3.0 x 10⁹ GC/g, about 3.5 x 10⁹ GC/g, about 4.0 x 10^{9 GC/g, about 4.5 x 10 9GC} /g, about 5.0 x 10⁹ GC/g, about 5.5 x 10⁹ GC/g, about 6.0 x 10⁹ GC/g, about 6.5 x 10⁹ GC/g, about 7.0 x 10⁹ GC/g, about 7.5 x 10⁹ GC/g, about 8.0 x 10 9 GC/g, about 8.5 x 10⁹ GC/g, about 9.0 x 10⁹ GC/g, about¹⁰ 10 GC/g, about 9.5x10⁹ GC/g, about 1.0x10¹⁰ GC/g, about 1.5x10¹⁰ GC/g, about 2.0x10¹⁰ GC/g, about 2.5x10¹⁰ GC/g, about 3.0x10¹⁰ GC/g, about 3.5x10¹⁰ GC/g, about 4.0x10¹⁰ GC/g, about 4.5x10¹⁰ GC/g, about 5.0x10¹⁰ GC/g, about 5.5x10¹⁰ GC/g, about 6.0x10¹⁰ GC/g, about 6.5x10¹⁰ GC/g, about 7.0x10¹⁰ GC/g, about 7.5x10¹⁰ GC/g, about 8.0x10¹⁰ GC/g 11 GC/g, about 8.5x10¹⁰ GC/g, about 9.0x10¹⁰ GC/g, about 9.5x10¹⁰ GC/g, about 1.0x10¹¹ GC/g, about 1.5x10¹¹ GC/g, about 2.0x10¹¹ GC/g, about 2.5x10¹¹ GC/g, about 3.0x10¹¹ GC/g, about 3.5x10¹¹ GC/g, about 4.0x10¹¹ GC/g, about 4.5x10¹¹ GC/g, about 5.0x10¹¹ GC/g, about 5.5x10¹¹ GC/g, about 6.0x10¹¹ GC/g, about 6.5x10¹¹ GC/g, about 7.0x10¹¹ 12 GC/g, about 7.5x10¹¹ GC/g, about 8.0x10¹¹ GC/g, about 8.5x10¹¹ GC/g, about 9.0x10¹¹ GC/g, about 9.5x10¹¹ GC/g, about 1.0x10¹² GC/g, about 1.5x10¹² GC/g, about 2.0x10¹² GC/g, about 2.5x10¹² GC/g, about 3.0x10¹² GC/g, about 3.5x10¹² GC/g, about 4.0x10¹² GC/g, about 4.5x10¹² GC/g, about 5.0x10¹² GC/g, about 5.5x10¹² GC/g, about 6.0x10¹² 13 GC/g, about 2.0x10¹³ GC/g, about 2.5x10¹³ GC/g, about 3.0x10¹³ GC/g, about 3.5x10¹³ GC/g, about 4.0x10¹³ GC/g, about 4.5x10 13 GC/g, about 5.0x10¹³ GC/g, about 5.0x10¹³ GC/g, about 6.5x10 12 GC/g, about 7.0x10¹² GC/g, about 7.5x10¹² GC/g, about 8.0x10 12 GC/g, about 8.5x10 12 GC/g, about 9.0x10 12 GC/g, about 9.5x10¹² GC/g, about 1.0x10¹³ GC/g, about 1.5x10 13 GC/g, about 2.0x10¹³ GC/g, about 2.5x10¹³ GC/g, about 3.0x10 13 GC/g, about 3.5x10 13 GC/g, about 4.0x10¹³ GC/g, about 4.5x10¹³ GC/g, about⁵ 13 GC/g, about 5.5x10¹³ GC/g, about 6.0x10¹³ GC/g, about 6.5x10¹³ GC/g, about 7.0x10¹³ GC/g, about 7.5x10¹³ GC/g, about 8.0x10¹³ GC/g, about 8.5x10¹³ GC/g, about 9.0x10¹³ GC/g, about 9.5x10¹³ GC/g, or about 1.0x10¹⁴ GC/g brain mass.

在某些实施例中，本文提供的组合物与免疫抑制剂联合施用。目前，用于这种联合治疗的免疫抑制剂包括但不限于糖皮质激素、类固醇、抗代谢物、T细胞抑制剂、大环内酯类(例如，雷帕霉素(rapamycin)或雷帕霉素类似物(rapalog))和细胞抑制剂，包括烷化剂、抗代谢物、细胞毒性抗生素、抗体或对免疫亲和素具有活性的药剂。免疫抑制剂可以包括氮芥(nitrogenmustard)、亚硝基脲、铂化合物、甲氨蝶呤(methotrexate)、硫唑嘌呤(azathioprine)、巯嘌呤(mercaptopurine)、氟尿嘧啶(fluorouracil)、放线菌素D(dactinomycin)、蒽环霉素(anthracycline)、丝裂霉素(mitomycin)C、博来霉素(bleomycin)、光神霉素(mithramycin)、IL-2受体-(CD25-)或CD3-定向抗体、抗IL-2抗体、环孢霉素(ciclosporin)、他克莫司(tacrolimus)、西罗莫司(sirolimus)、IFN-β、IFN-γ、阿片样药物(opioid)或TNF-α(肿瘤坏死因子-α)结合剂。在某些实施例中，免疫抑制治疗可以在基因疗法施用前0、1、2、7或更多天开始。这种疗法可以包括在同一天联合施用两种或更多种药物(例如，泼尼松(prednisone)、霉酚酸酯(MMF)和/或西罗莫司(即，雷帕霉素))。这些药物中的一种或多种可以在基因疗法施用后以相同剂量或调整剂量继续施用。In certain embodiments, the compositions provided herein are co-administered with an immunosuppressant. At present, the immunosuppressant used for this combination therapy includes but is not limited to glucocorticoids, steroids, antimetabolites, T cell inhibitors, macrolides (e.g., rapamycin or rapamycin analogs (rapalog)) and cytostatics, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies or agents active against immunophilins. Immunosuppressants can include nitrogen mustard, nitrosourea, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor-(CD25-) or CD3-directed antibodies, anti-IL-2 antibodies, ciclosporin, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids or TNF-α (tumor necrosis factor-α) binding agents. In certain embodiments, immunosuppressive therapy can be started 0, 1, 2, 7 or more days before gene therapy is administered. This therapy may include co-administering two or more drugs (e.g., prednisone, mycophenolate mofetil (MMF), and/or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may continue to be administered at the same dose or adjusted dose after the gene therapy is administered.

在某些实施例中，本文提供的rAAV与如酶替代疗法、伴侣疗法、底物减少疗法(例如，赛诺菲-健赞(Sanofi-Genzyme)和宜道赛(Idorsia))的疗法(联合治疗)联合施用，和/或与减少输注相关反应的机会的抗组胺药或其它药物联合施用。在某些实施例中，联合治疗是功能性hGLA蛋白(例如赛诺菲-健赞；Shire；基于植物的ERT)或hGLA的稳定形式，如本文提供的或如2019年10月10日提交的PCT/US2019/05567(其通过引用并入本文)中所述的。施用可以是口服或通过静脉内输注给门诊病人，并且可以包括适于每日、每隔一日、每周、每两周(例如，0.2mg/kg体重)，每月或每两月施用的剂量。在某些实施例中，联合治疗是伴侣疗法(例如Galafold(米加司他，以胶囊形式口服递送)，爱美医疗公司)。联合治疗的适当治疗有效剂量由治疗临床医师选择，并且包括约1μg/kg至约500mg/kg、约10mg/kg至约100mg/kg、约20mg/kg至约100mg/kg和大约20mg/kg至大约50mg/kg。在一些实施例中，合适的治疗剂量选自例如0.5、0.75、1、5、10、15、20、30、40、50、60、70、100、150、200、250、300、400或500mg/kg。In certain embodiments, the rAAV provided herein is administered in combination with a therapy such as enzyme replacement therapy, chaperone therapy, substrate reduction therapy (e.g., Sanofi-Genzyme and Idorsia) (combination therapy), and/or with an antihistamine or other drug that reduces the chance of an infusion-related reaction. In certain embodiments, the combination therapy is a functional hGLA protein (e.g., Sanofi-Genzyme; Shire; Plant-based ERT) or a stable form of hGLA, as provided herein or as described in PCT/US2019/05567 filed on October 10, 2019 (which is incorporated herein by reference). Administration can be oral or by intravenous infusion to outpatients, and can include dosages suitable for daily, every other day, weekly, every two weeks (e.g., 0.2 mg/kg body weight), monthly or bimonthly administration. In certain embodiments, the combination therapy is a companion therapy (e.g., Galafold (migalastat, delivered orally in capsule form), Aimee Medical). The appropriate therapeutically effective dose of the combination therapy is selected by the treating clinician and includes about 1 μg/kg to about 500 mg/kg, about 10 mg/kg to about 100 mg/kg, about 20 mg/kg to about 100 mg/kg, and about 20 mg/kg to about 50 mg/kg. In some embodiments, a suitable therapeutic dose is selected from, for example, 0.5, 0.75, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 100, 150, 200, 250, 300, 400, or 500 mg/kg.

在某些实施例中，根据本文所述的方法治疗新生婴儿(3月龄或更小)。在某些实施例中，根据本文所述的方法治疗3月龄至9月龄的婴儿。在某些实施例中，根据本文所述的方法治疗9月龄至36月龄的儿童。在某些实施例中，根据本文所述的方法治疗3岁至12岁的儿童。在某些实施例中，根据本文所述的方法治疗12岁至18岁的儿童。在某些实施例中，根据本文所述的方法治疗18岁或以上的成人。In certain embodiments, newborn infants (3 months of age or younger) are treated according to the methods described herein. In certain embodiments, infants 3 months to 9 months of age are treated according to the methods described herein. In certain embodiments, children 9 months to 36 months of age are treated according to the methods described herein. In certain embodiments, children 3 years to 12 years of age are treated according to the methods described herein. In certain embodiments, children 12 years to 18 years of age are treated according to the methods described herein. In certain embodiments, adults 18 years of age or older are treated according to the methods described herein.

在一个实施例中，患有法布里病的患者是年龄至少约3个月至小于12个月的男性或女性。在另一实施例中，患有法布里病的患者是男性或女性，年龄为至少约6岁至高达18岁。在其它实施例中，受试者可以是年长或年幼的，并且可以是男性或女性。In one embodiment, the patient with Fabry disease is a male or female at least about 3 months to less than 12 months of age. In another embodiment, the patient with Fabry disease is a male or female at least about 6 years of age to up to 18 years of age. In other embodiments, the subject can be older or younger, and can be male or female.

应当理解，本文所述方法中的组合物旨在应用于说明书中描述的其它组合物、方案、方面、实施例和方法。It is to be understood that the compositions in the methods described herein are intended to be applicable to other compositions, schemes, aspects, embodiments, and methods described in the specification.

7.试剂盒7. Test kit

在某些实施例中，提供了一种试剂盒，其包括悬浮在制剂(任选冷冻的)中的浓缩载体、任选的稀释缓冲液，以及静脉内、鞘内、脑室内或脑池内施用所需的装置和组件。在一个实施例中，试剂盒提供足够的缓冲液以允许注射。这种缓冲液可以允许浓缩载体以约1∶1到1∶5的比例稀释，或者更高。这种试剂盒可以包括额外的非基于载体的活性组分，其中使用联合疗法和/或抗组胺药、免疫调节剂等。在其它实施例中，包括较高或较低量的缓冲液或无菌水以允许治疗临床医师进行剂量滴定和其它调整。在其它实施例中，装置的一个或多个组件包括在试剂盒中。合适的稀释缓冲液是可用的，如盐水、磷酸盐缓冲盐水(PBS)或甘油/PBS。In certain embodiments, a kit is provided, which includes a concentrated carrier suspended in a formulation (optionally frozen), an optional dilution buffer, and the devices and components required for intravenous, intrathecal, intraventricular or intracisternal administration. In one embodiment, the kit provides enough buffer to allow injection. This buffer can allow the concentrated carrier to be diluted at a ratio of about 1:1 to 1:5, or higher. This kit can include additional non-carrier-based active components, in which combination therapy and/or antihistamines, immunomodulators, etc. are used. In other embodiments, a higher or lower amount of buffer or sterile water is included to allow the treating clinician to perform dose titration and other adjustments. In other embodiments, one or more components of the device are included in the kit. Suitable dilution buffers are available, such as saline, phosphate buffered saline (PBS) or glycerol/PBS.

应当理解，本文所述试剂盒中的组合物旨在应用于说明书中描述的其它组合物、方案、方面、实施例和方法。It is to be understood that the compositions in the kits described herein are intended to be applied to other compositions, schemes, aspects, embodiments and methods described in the specification.

8.装置8. Installation

在一个方面，本文提供的载体可以通过例如WO 2017/136500中所述的方法和/或装置鞘内施用，该专利通过引用整体并入本文。替代地，可以选择其它装置和方法。总之，该方法包含以下步骤：将脊椎穿刺针推进到患者的小脑延髓池内，将一段柔性管连接到脊椎穿刺针的近端毂，以及将阀的输出端口连接到柔性管的近端，并且在所述推进和连接步骤之后以及在允许管用患者的脑脊液进行自灌注之后，将含有一定量等渗溶液的第一容器连接到阀的冲洗入口，然后将含有一定量药物组合物的第二容器连接到阀的载体入口。在将第一容器和第二容器连接到阀之后，打开在阀的载体入口和出口之间的流体流动路径，并且通过脊椎穿刺针将药物组合物注射到患者体内，并且在注射药物组合物之后，打开通过阀的冲洗入口和出口的流体流动路径，并且将等渗溶液注射到脊椎穿刺针中，以将药物组合物冲洗到患者体内。该方法和该装置可以各自任选地用于鞘内递送本文提供的组合物。替代地，其它方法和装置可用于这种鞘内递送。In one aspect, the carrier provided herein can be administered intrathecally by the method and/or device described in, for example, WO 2017/136500, which is incorporated herein by reference in its entirety. Alternatively, other devices and methods can be selected. In summary, the method comprises the following steps: advancing a spinal puncture needle into the patient's cerebellomedullary cisterna, connecting a section of flexible tubing to the proximal hub of the spinal puncture needle, and connecting the output port of the valve to the proximal end of the flexible tube, and after the advancement and connection steps and after allowing the tube to be self-perfused with the cerebrospinal fluid of the patient, connecting a first container containing a certain amount of isotonic solution to the flushing inlet of the valve, and then connecting a second container containing a certain amount of pharmaceutical composition to the carrier inlet of the valve. After the first container and the second container are connected to the valve, the fluid flow path between the carrier inlet and outlet of the valve is opened, and the pharmaceutical composition is injected into the patient's body by the spinal puncture needle, and after the injection of the pharmaceutical composition, the fluid flow path through the flushing inlet and outlet of the valve is opened, and the isotonic solution is injected into the spinal puncture needle to flush the pharmaceutical composition into the patient's body. The method and the device can each be optionally used for intrathecal delivery of the composition provided herein. Alternatively, other methods and devices may be used for such intrathecal delivery.

应当理解，本文所述装置中的组合物旨在应用于说明书中描述的其它组合物、方案、方面、实施例和方法。It should be understood that the compositions in the devices described herein are intended to be applicable to other compositions, schemes, aspects, embodiments and methods described in the specification.

实例Examples

现在参考以下实例描述本发明。提供这些实例仅用于说明的目的，本发明决不应解释为限于这些实例，而是应解释为包括由于本文提供的教导而变得明显的任何和所有变化。The present invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the present invention should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.

实例1：用于治疗法布里病的rAAVhu68.hGLAExample 1: rAAVhu68.hGLA for the treatment of Fabry disease

将编码hGLA的工程化序列克隆到含有CB7启动子(巨细胞病毒立即早期增强子和鸡β-肌动蛋白启动子的杂交体)、鸡β-肌动蛋白内含子(CI)、WPRE和兔β珠蛋白(rBG)聚腺苷酸化序列的表达构建体中。表达构建体的侧翼为AAV2反向末端重复序列，AAVhu68反式质粒用于衣壳化。The engineered sequence encoding hGLA was cloned into an expression construct containing the CB7 promoter (a hybrid of the cytomegalovirus immediate early enhancer and the chicken β-actin promoter), the chicken β-actin intron (CI), WPRE, and the rabbit β-globin (rBG) polyadenylation sequence. The expression construct was flanked by AAV2 inverted terminal repeats and the AAVhu68 trans plasmid was used for encapsidation.

rAAVhu68.hGLA是通过用编码侧翼为AAVITR的转基因盒的AAV顺式质粒、编码AAV2rep和AAVhu68 cap基因的AAV反式质粒(pAAV2/hu68.KanR)和辅助腺病毒质粒(pAdΔF6.KanR)三重质粒转染HEK293细胞而产生的。rAAVhu68.hGLA was produced by triple plasmid transfection of HEK293 cells with an AAV cis plasmid encoding the transgene cassette flanked by AAV ITR, an AAV trans plasmid encoding the AAV2 rep and AAVhu68 cap genes (pAAV2/hu68.KanR), and a helper adenovirus plasmid (pAdΔF6.KanR).

A.AAV顺式质粒A. AAV cis plasmid

载体基因组(SEQ ID NO：6)的图谱如图1所示。载体基因组含有以下序列元件：The map of the vector genome (SEQ ID NO: 6) is shown in Figure 1. The vector genome contains the following sequence elements:

反向末端重复序列(ITR)：ITR是衍生自AAV2(130bp，基因库：NC_001401)的相同的反向互补序列，其侧接载体基因组的所有组分。当AAV和腺病毒辅助功能以反式提供时，ITR作为载体DNA复制的起点和载体基因组的包装信号。因此，ITR序列代表载体基因组复制和包装所需的唯一顺式序列。Inverted terminal repeats (ITRs): ITRs are identical reverse complementary sequences derived from AAV2 (130 bp, GenBank: NC_001401) that flank all components of the vector genome. When AAV and adenovirus helper functions are provided in trans, ITRs serve as the origin of vector DNA replication and packaging signal for the vector genome. Therefore, ITR sequences represent the only cis sequences required for vector genome replication and packaging.

CB7启动子：该启动子由CMV IE增强子和鸡β-肌动蛋白启动子之间的杂交体组成。CB7 Promoter: This promoter consists of a hybrid between the CMV IE enhancer and the chicken β-actin promoter.

人巨细胞病毒立即早期(CMVIE)增强子：这种从人源CMV(基因库：K03104.1)获得的增强子序列增加了下游转基因的表达。Human cytomegalovirus immediate-early (CMVIE) enhancer: This enhancer sequence obtained from human CMV (GenBank: K03104.1) increases the expression of downstream transgenes.

鸡β-肌动蛋白(CB)启动子：选择这种普遍存在的启动子(基因库：X00182.1)来驱动任何细胞类型中的转基因表达。Chicken β-actin (CB) promoter: This ubiquitous promoter (GenBank: X00182.1) was selected to drive transgene expression in any cell type.

鸡β-肌动蛋白内含子：杂合内含子由鸡β-肌动蛋白剪接供体(973bp，基因库：X00182.1)和兔β-珠蛋白剪接受体元件组成。内含子被转录，但通过剪接从成熟mRNA中去除，将其任一侧的序列集合在一起。已经表明表达盒中内含子的存在促进mRNA从细胞核转运到细胞质，从而增强用于翻译的mRNA的稳定水平的积累。这是旨在提高基因表达水平的基因载体中的共同特征。Chicken β-actin intron: The hybrid intron consists of the chicken β-actin splice donor (973 bp, GenBank: X00182.1) and the rabbit β-globin splice acceptor elements. The intron is transcribed but removed from the mature mRNA by splicing, bringing together the sequences on either side of it. The presence of an intron in the expression cassette has been shown to promote transport of mRNA from the nucleus to the cytoplasm, thereby enhancing the accumulation of a steady level of mRNA for translation. This is a common feature in gene vectors designed to increase gene expression levels.

编码序列：工程化cDNA(SEQ ID NO：4)，其编码hGLA(SEQ ID NO：7)，在位置233和359处具有半胱氨酸残基(D233C.I359C)(431个氨基酸)。Coding sequence: Engineered cDNA (SEQ ID NO: 4) encoding hGLA (SEQ ID NO: 7) with cysteine residues at positions 233 and 359 (D233C.I359C) (431 amino acids).

土拨鼠肝炎病毒转录后调节元件(WPRE)：将来自土拨鼠肝炎病毒(WHV)的顺式作用RNA元件插入polyA信号上游的编码序列的3′非翻译区。WPRE是一种源于嗜肝DNA病毒的序列，并且先前已被用作病毒基因载体中的顺式作用调节模块以实现足够水平的转基因产物表达并在制造期间提高病毒滴度。认为WPRE通过改善转录终止和增强3′端转录加工来增加转基因产物表达，从而增加聚腺苷酸化转录物的量和polyA尾的大小，并导致更多可用于翻译的转基因mRNA。包含在顺式质粒中的WPRE是在土拨鼠肝炎病毒X蛋白(WHX)蛋白开放阅读框(ORF)的推定启动子区中含有五个点突变，以及在WHX蛋白ORF的起始密码子中含有额外的点突变(突变为TTG的ATG)的突变版本。基于用含有WPRE mut6-GFP融合构建体(Zanta-Boussif等人，2009)的慢病毒转导的各种人细胞系的灵敏流式细胞术分析，认为该突变WPRE(称为mut6)足以消除截短的WHX蛋白的表达。Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE): A cis-acting RNA element from the Woodchuck Hepatitis Virus (WHV) is inserted into the 3′ untranslated region of the coding sequence upstream of the polyA signal. The WPRE is a sequence derived from a hepadnavirus and has been previously used as a cis-acting regulatory module in viral gene vectors to achieve adequate levels of transgene product expression and to increase viral titers during manufacturing. The WPRE is thought to increase transgene product expression by improving transcription termination and enhancing 3′ end transcription processing, thereby increasing the amount of polyadenylated transcripts and the size of the polyA tail, and resulting in more transgene mRNA available for translation. The WPRE contained in the cis plasmid is a mutant version containing five point mutations in the putative promoter region of the Woodchuck Hepatitis Virus X protein (WHX) protein open reading frame (ORF), as well as an additional point mutation (ATG to TTG) in the start codon of the WHX protein ORF. Based on sensitive flow cytometric analysis of various human cell lines transduced with lentivirus containing a WPRE mut6-GFP fusion construct (Zanta-Boussif et al., 2009), it was suggested that this mutant WPRE (termed mut6) is sufficient to abrogate the expression of truncated WHX proteins.

WPRE是一种源于嗜肝DNA病毒的序列，并且先前已用作病毒基因载体中的顺式作用调节模块以实现足够水平的转基因产物表达并在制造期间提高病毒滴度。WPRE is a sequence derived from a hepadnavirus and has been previously used as a cis-acting regulatory module in viral gene vectors to achieve adequate levels of transgene product expression and to increase viral titers during manufacturing.

兔β-珠蛋白聚腺苷酸化信号(rBG PolyA)：rBG PolyA信号(127bp，基因库：V00882.1)促进顺式转基因mRNA的有效聚腺苷酸化。该元件用作转录终止的信号，在新生转录物的3′端发生特异性切割事件，并添加长聚腺苷酸尾。Rabbit β-globin polyadenylation signal (rBG PolyA): The rBG PolyA signal (127 bp, GenBank: V00882.1) promotes efficient polyadenylation of cis-transgenic mRNA. This element serves as a signal for transcription termination, with a specific cleavage event occurring at the 3′ end of the nascent transcript and the addition of a long polyadenylic acid tail.

B.反式质粒：pAAV2/1.KanR(p0068)B. Trans-plasmid: pAAV2/1.KanR (p0068)

AAV2/hu68反式质粒是pAAV2/hu68.KanR(p0068)。pAAV2/hu68.KanR质粒长度为8030bp，并编码AAV载体基因组复制和包装所需的四种野生型AAV2复制酶(Rep)蛋白。pAAV2/hu68.KanR质粒还编码三种WTAAVhu68病毒体蛋白衣壳(Cap)蛋白，其组装成AAV血清型hu68的病毒体外壳以容纳AAV载体基因组。The AAV2/hu68 trans plasmid is pAAV2/hu68.KanR (p0068). The pAAV2/hu68.KanR plasmid is 8030 bp in length and encodes four wild-type AAV2 replicase (Rep) proteins required for AAV vector genome replication and packaging. The pAAV2/hu68.KanR plasmid also encodes three wild-type AAV2 virion protein capsid (Cap) proteins, which assemble into the virion shell of AAV serotype hu68 to accommodate the AAV vector genome.

C.腺病毒辅助质粒：pAdDeltaF6(KanR)C. Adenovirus helper plasmid: pAdDeltaF6 (KanR)

腺病毒辅助质粒pAdDeltaF6(KanR)大小为15,770bp。该质粒含有对AAV复制重要的腺病毒基因组区域；即，E2A、E4和VA RNA(腺病毒E1功能由HEK293细胞提供)。然而，质粒不含有其它腺病毒复制或结构基因。质粒不含对复制关键的顺式元件，如腺病毒ITR；因此，预期不会生成感染性腺病毒。质粒来源于Ad5的E1、E3缺失的分子克隆(pBHG10，一种基于pBR322的质粒)。将缺失引入Ad5以消除不必要的腺病毒基因的表达并将腺病毒DNA的量从32kb减少至12kb。最后，用卡那霉素(kanamycin)抗性基因替换氨苄青霉素(ampicillin)抗性基因以产生pAdeltaF6(KanR)。保留在该质粒中的E2、E4和VAI腺病毒基因以及存在于HEK293细胞中的E1对于AAV载体生产是必需的。The adenovirus helper plasmid pAdDeltaF6 (KanR) is 15,770 bp in size. This plasmid contains regions of the adenovirus genome that are important for AAV replication; i.e., E2A, E4, and VA RNA (adenovirus E1 function is provided by HEK293 cells). However, the plasmid does not contain other adenovirus replication or structural genes. The plasmid does not contain cis elements that are critical for replication, such as adenovirus ITRs; therefore, infectious adenovirus is not expected to be generated. The plasmid is derived from a molecular clone (pBHG10, a pBR322-based plasmid) of E1 and E3 deletions of Ad5. Deletions are introduced into Ad5 to eliminate the expression of unnecessary adenovirus genes and reduce the amount of adenovirus DNA from 32 kb to 12 kb. Finally, the ampicillin resistance gene is replaced with a kanamycin resistance gene to produce pAdeltaF6 (KanR). The E2, E4 and VAI adenoviral genes retained in this plasmid and the E1 present in HEK293 cells are essential for AAV vector production.

实例2：方法Example 2: Method

转基因产物表达-细胞分布Transgenic product expression-cellular distribution

为了表征IV施用AAVhu69.hGLA载体后转导和转基因产物表达的分布并将其与观察到的疾病表型的组织学改善相关联，评估了mRNA和蛋白质定位。选择肾、DRG和心脏组织进行评估，因为它们是用于治疗法布里病的疾病相关靶组织。在小鼠和NHP中通过原位杂交(ISH)评估人GLA mRNA。在小鼠和NHP中通过免疫组织化学(IHC)或免疫荧光(1F)评估人GLA蛋白。选择小鼠和NHP中的取样时间点以捕获转基因产物表达的预期稳定平台期间的表达。To characterize the distribution of transduction and transgene product expression after IV administration of AAVhu69.hGLA vector and correlate it with the histological improvement of the observed disease phenotype, mRNA and protein localization were evaluated. Kidney, DRG and heart tissues were selected for evaluation because they are disease-related target tissues for the treatment of Fabry disease. Human GLA mRNA was evaluated by in situ hybridization (ISH) in mice and NHPs. Human GLA protein was evaluated by immunohistochemistry (IHC) or immunofluorescence (IF) in mice and NHPs. Sampling time points in mice and NHPs were selected to capture expression during the expected stable platform of transgene product expression.

转基因产物表达-功能活性Transgenic product expression-functional activity

为了评估通过ISH和IHC观察到的转基因产物在小鼠和NHP中是否有功能，进行了GLA酶活性测定。选择肾、心脏、肝和DRG组织是因为它们是用于治疗法布里病的疾病相关靶组织(肾、心脏)和/或在IV基因治疗后易于转导的组织(肝、心脏、DRG)。转基因产物的表达在小鼠的DRG中由于它们的小尺寸而未被评估，而所有其它组织在小鼠和NHP中被评估。选择小鼠和NHP中的取样时间点以捕获稳定的平台期转基因表达。GLA活性测定不能区分人GLA转基因产物和内源小鼠或NHPGLA，因此在基线时在未处理的动物中可以预期一些背景活性。In order to assess whether the transgenic product observed by ISH and IHC has function in mice and NHP, GLA enzyme activity assay was carried out.Kidney, heart, liver and DRG tissues are selected because they are disease-related target tissues (kidney, heart) for the treatment of Fabry disease and/or are easy to transduce tissues (liver, heart, DRG) after IV gene therapy.The expression of transgenic product is not assessed in the DRG of mice due to their small size, and all other tissues are assessed in mice and NHP.The sampling time point in mice and NHP is selected to capture stable plateau transgenic expression.GLA activity assay can not distinguish between people's GLA transgenic product and endogenous mice or NHPGLA, therefore some background activities can be expected in untreated animals at baseline.

热感觉功能Thermal Sensation Function

进行热板测定是因为其测量小鼠中的热感觉缺陷，据信其类似于法布里病患者中描述的继发于DRG神经元溶酶体贮积和功能障碍的触觉、疼痛和热感缺陷。潜伏期应答的降低表明法布里病表型的改善。The hot plate assay was performed because it measures thermal sensory deficits in mice that are believed to be similar to the touch, pain, and thermal sensory deficits described in Fabry disease patients secondary to lysosomal storage and dysfunction of DRG neurons. A decrease in latency response indicates an improvement in the Fabry disease phenotype.

肾功能Renal function

对BUN、尿渗透压和尿量进行了评估，因为它们是肾功能的生物标志物。BUN水平的降低表明法布里病表型的改善。尿渗透压的增加将表明由于增加的肾功能而增加的浓缩尿的能力，这代表法布里病表型的改善。尿量的减少表明法布里病表型的改善。BUN, urine osmolality, and urine volume were assessed because they are biomarkers of renal function. A decrease in BUN levels indicates an improvement in the Fabry disease phenotype. An increase in urine osmolality would indicate an increased ability to concentrate urine due to increased renal function, which represents an improvement in the Fabry disease phenotype. A decrease in urine volume indicates an improvement in the Fabry disease phenotype.

溶酶体贮积(组织上的GL-3免疫组织化学)Lysosomal storage (GL-3 immunohistochemistry on tissues)

GLA酶缺乏导致酶的毒性底物GL-3的积累。因此，对DRG和肾进行GL-3的IHC，因为它们是在经典(Gla KO)和加重(Gla KO/TgG3S)法布里病小鼠模型中可再现地显示显著储存的器官，并且是法布里病患者中病理学的靶器官(分别引起神经性疼痛和致命性肾衰竭)。对心脏进行GL-3 IHC，因为加重的(Gla KO/TgG3S)法布里病小鼠也在该器官中表现出储存。减少的GL-3储存表明法布里病表型的改善。GL-3 IHC切片也用苏木精和曙红(H&E)染色以更好地可视化组织形态并检测潜在的不良治疗相关发现。GLA enzyme deficiency causes the accumulation of toxic substrate GL-3 of enzyme.Therefore, GL-3 IHC is carried out to DRG and kidney, because they are organs that can be reproducibly shown to be significantly stored in classical (Gla KO) and aggravated (Gla KO/TgG3S) Fabry disease mouse model, and are the target organs of pathology in Fabry disease patients (causing neuropathic pain and fatal renal failure respectively). GL-3 IHC is carried out to heart, because aggravated (Gla KO/TgG3S) Fabry disease mice also show storage in this organ. Reduced GL-3 storage shows the improvement of Fabry disease phenotype. GL-3 IHC sections are also stained with hematoxylin and eosin (H&E) to better visualize tissue morphology and detect potential adverse treatment-related findings.

溶酶体贮积(通过LC-MS/MS定量GL-3和Lvso-Gb₃)Lysosomal storage (quantification of GL-3 and Lvso-Gb₃ by LC-MS/MS)

通过液相色谱-串联质谱(LC-MS/MS)对组织中GL-3的储存进行定量，对血浆或血清中的lyso-Gb₃进行定量。由于GL-3是GLA酶的主要底物，并且有直接证据表明底物累积的严重程度与法布里病的严重程度之间存在关系，因此对靶器官中的储存进行定量。GL-3储存的减少表明法布里病表型的改善。GL-3 storage in tissues is quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and lyso-Gb₃ in plasma or serum is quantified. Because GL-3 is the major substrate for the GLA enzyme and there is direct evidence of a relationship between the severity of substrate accumulation and the severity of Fabry disease, storage in target organs is quantified. A reduction in GL-3 storage indicates an improvement in the Fabry disease phenotype.

实例3：法布里病的加重(Gla KO/TgG3S)和经典(Gla KO)小鼠模型的自然史研究Example 3: Natural history study of the aggravated (Gla KO/TgG3S) and classic (Gla KO) mouse models of Fabry disease

进行自然史研究以表征法布里病加重的小鼠模型(Gla KO/TgG3S)的疾病进展并定义疗效研究的最佳药理学终点和治疗窗。A natural history study was performed to characterize disease progression in a mouse model of Fabry disease exacerbations (Gla KO/TgG3S) and to define optimal pharmacological endpoints and therapeutic windows for efficacy studies.

在出生时，本研究入组了41只小鼠，包括不带有TgG3S等位基因的Gla WT雄性或Gla^+/-杂合子雌性(对照；5只雄性和6只雌性)、不带有TgG3S等位基因的Gla KO(Gla^-/-；5只雄性和5只雌性)、带有TgG3S的一个等位基因的Gla WT雄性或Gla^+/-杂合子雌性(TgG3S⁺；5只雄性和5只雌性)和带有TgG3S的一个等位基因的Gla KO(Gla KO/TgG3S；5只雄性和5只雌性)。定期评估体重、热板性能、血清BUN水平和尿渗透压。在36周龄时进行尸检，这接近于该模型的公布的人道终点。在尸检时收集大脑、脊髓、DRG、心脏、肾脏、肝脏、皮肤、小肠和大肠用于组织学和GL-3储存的评估(GL-3IHC和通过LC-MS/MS定量)。At birth, 41 mice were enrolled in this study, including Gla WT males or Gla^+/- heterozygous females without the TgG3S allele (control; 5 males and 6 females), Gla KO without the TgG3S allele (Gla^-/- ; 5 males and 5 females), Gla WT males or Gla^+/- heterozygous females with one allele of TgG3S (TgG3S⁺ ; 5 males and 5 females), and Gla KO with one allele of TgG3S (Gla KO/TgG3S; 5 males and 5 females). Body weight, hot plate performance, serum BUN levels, and urine osmolality were assessed regularly. Necropsy was performed at 36 weeks of age, which is close to the published humane endpoint of this model. Brain, spinal cord, DRG, heart, kidney, liver, skin, small intestine, and large intestine were collected at necropsy for histology and assessment of GL-3 storage (GL-3 IHC and quantification by LC-MS/MS).

研究设计：加重法布里Gla^-/-/TgG3S⁺小鼠模型中的自然史研究Study Design: Natural History Study in an Aggravated Fabry Gla^-/- /TgG3S⁺ Mouse Model

^a收集组织用于评估组织病理学。^a Tissues were collected for histopathological evaluation.

缩写：BUN，血尿氮；Gla，α-半乳糖苷酶A；TgG3S，人Gb3合酶-转基因的。Abbreviations: BUN, blood urine nitrogen; Gla, α-galactosidase A; TgG3S, human Gb3 synthase-transgenic.

除了两只Gla KO/TgG3S小鼠在33周和35周时由于疾病相关的体重减轻而被安乐死之外，所有小鼠都存活至预定的第36周尸检。All mice survived until the scheduled 36-week necropsy, except for two Gla KO/TgG3S mice that were euthanized at 33 and 35 weeks due to disease-related weight loss.

对照、GlaKO和TgG3S小鼠在整个研究的每个时间点增加体重(图10A和图10B)。相反，GlaKO/TgG3S小鼠的体重在18周时达到峰值(雄性为24.4g，雌性为21.5g)，此后小鼠开始体重减轻直至尸检。Control, GlaKO and TgG3S mice gained weight at every time point throughout the study (Figure 10A and Figure 10B). In contrast, the body weight of GlaKO/TgG3S mice peaked at 18 weeks (24.4 g for males and 21.5 g for females), after which the mice began to lose weight until necropsy.

在整个研究中，雄性和雌性TgG3S小鼠表现出与性别匹配的对照动物相似的热板潜伏期，表明正常的感觉应答。从25周龄到研究结束，与性别匹配的对照相比，雄性和雌性GlaKO小鼠表现出稍长的干均热板潜伏期，表明感觉应答稍有降低。相反，从25周龄到研究结束，雄性GlaKO/TgG3S小鼠表现出比对照或GlaKO小鼠(雄性或雌性)显著更长的平均潜伏期应答，表明雄性GlaKO/TgG3S小鼠比雄性和雌性GlaKO小鼠具有更严重的感觉缺陷。雌性GlaKO/TgG3S小鼠也表现出比对照小鼠稍长的热板潜伏期应答，但潜伏期与雌性GlaKO小鼠相似，表明两种雌性法布里病小鼠模型的感觉缺陷相似(图11A和图11B)。Throughout the study, male and female TgG3S mice showed similar hot plate latencies to sex-matched control animals, indicating normal sensory responses. From 25 weeks of age to the end of the study, male and female GlaKO mice showed slightly longer dry hot plate latencies compared to sex-matched controls, indicating that sensory responses were slightly reduced. On the contrary, from 25 weeks of age to the end of the study, male GlaKO/TgG3S mice showed significantly longer average latency responses than controls or GlaKO mice (male or female), indicating that male GlaKO/TgG3S mice had more severe sensory defects than male and female GlaKO mice. Female GlaKO/TgG3S mice also showed slightly longer hot plate latency responses than control mice, but the latency was similar to that of female GlaKO mice, indicating that sensory defects in two female Fabry disease mouse models were similar (Figures 11A and 11B).

在整个研究中，雄性和雌性TgG3S和Gla KO小鼠表现出与其性别匹配的对照相似的血清BUN水平，这表明肾功能正常。相反，与性别匹配的对照相比，25周龄的雄性和雌性Gla KO/TgG3S小鼠均表现出升高的BUN水平。在研究过程中，雄性和雌性的BUN水平普遍升高，表明肾功能下降。在整个研究中，雄性和雌性Gla KO/TgG3S小鼠的BUN水平大致相似(图12A和图12B)。Throughout the study, male and female TgG3S and Gla KO mice showed serum BUN levels similar to their sex-matched controls, indicating normal renal function. In contrast, male and female Gla KO/TgG3S mice at 25 weeks of age showed elevated BUN levels compared to sex-matched controls. During the study, BUN levels in males and females generally increased, indicating decreased renal function. Throughout the study, BUN levels in males and females Gla KO/TgG3S mice were roughly similar (Figures 12A and 12B).

在研究中观察到尿渗透压的动物体内变异性。然而，从25周龄直到研究结束，雄性和雌性Gla KO/TgG3S小鼠通常表现出比性别匹配的对照和Gla KO小鼠更低的平均尿渗透压，表明由于肾功能降低而不能浓缩尿(图13A和图13B)。Intra-animal variability in urine osmolality was observed in the study. However, from 25 weeks of age until the end of the study, male and female Gla KO/TgG3S mice generally exhibited lower mean urine osmolality than sex-matched control and Gla KO mice, indicating an inability to concentrate urine due to reduced renal function (Figures 13A and 13B).

当通过IHC评估时，与Gla KO或WT/TgG3S小鼠相比，在雄性Gla KO/TgG3S小鼠中观察到更显著的GL-3在肾中的储存以及肾炎和肾小管坏死的继发性损伤(图14A)。与Gla KO小鼠相比，Gla KO/TgG3S小鼠中GL-3储存和继发性病理学的程度更大。在Gla KO/TgG3S小鼠的肾脏中，在肾小管和肾小球细胞中均观察到储存材料，而GL-3储存仅在Gla KO小鼠的肾小管中观察到。除了在肾小管和肾小球中有更多的储存之外，一些Gla KO/TgG3S小鼠在肾脏中呈现继发性炎性和退行性病变(肾小管变性、坏死和继发性间质单核肾炎)，这在任何Gla KO小鼠中均未见到。心脏在Gla KO小鼠中没有表现出任何GL-3储存或继发性损伤，在一些Gla KO/TgG3S动物中，在心肌细胞中表现出一些GL-3储存材料以及心肌细胞坏死和矿化。When evaluated by IHC, more significant storage of GL-3 in the kidney and secondary damage of nephritis and tubular necrosis were observed in male Gla KO/TgG3S mice compared with Gla KO or WT/TgG3S mice (Figure 14A). Compared with Gla KO mice, the degree of GL-3 storage and secondary pathology in Gla KO/TgG3S mice is greater. In the kidney of Gla KO/TgG3S mice, storage material is observed in both tubular and glomerular cells, while GL-3 storage is only observed in the tubular of Gla KO mice. In addition to having more storage in tubular and glomerular, some Gla KO/TgG3S mice present secondary inflammatory and degenerative lesions (tubular degeneration, necrosis and secondary interstitial mononuclear nephritis) in the kidney, which is not seen in any Gla KO mice. The hearts did not show any GL-3 storage or secondary injury in Gla KO mice, but showed some GL-3 storage material in cardiomyocytes as well as cardiomyocyte necrosis and mineralization in some Gla KO/TgG3S animals.

GL-3 IHC的定量证实，与Gla KO或WT/TgG3S小鼠相比，雄性Gla KO/TgG3S小鼠在整个肾脏中具有显著更高水平的GL-3储存，其中WT/TgG3S小鼠在3种小鼠模型中具有最低水平的GL-3储存(图14B)。Quantification of GL-3 IHC confirmed that male Gla KO/TgG3S mice had significantly higher levels of GL-3 storage throughout the kidney compared to Gla KO or WT/TgG3S mice, with WT/TgG3S mice having the lowest levels of GL-3 storage among the three mouse models ( FIG. 14B ).

当通过IHC评估时，雄性Gla KO/TgG3S小鼠在DRG感觉神经元中表现出显著的GL-3储存(图15A)。虽然雄性Gla KO小鼠也在DRG感觉神经元中表现出GL-3储存，但在雄性WT/TgGS3小鼠中观察到非常少的DRG GL-3储存。IHC染色的定量显示，与Gla KO或WT/TgG3S模型相比，Gla KO/TgG3S小鼠中DRG GL-3储存显著增加，WT/TgG3S小鼠表现出最低水平的DRGGL-3储存(图15B)。When evaluated by IHC, male Gla KO/TgG3S mice showed significant GL-3 storage in DRG sensory neurons (Figure 15A). Although male Gla KO mice also showed GL-3 storage in DRG sensory neurons, very little DRG GL-3 storage was observed in male WT/TgGS3 mice. Quantitative IHC staining showed that DRG GL-3 storage was significantly increased in Gla KO/TgG3S mice compared to Gla KO or WT/TgG3S models, and WT/TgG3S mice showed the lowest level of DRGGL-3 storage (Figure 15B).

通过LC-MS/MS对底物(血浆中的lyso-Gb₃，组织中的GL-3)的定量证实，与Gla KO小鼠相比，这些底物在加重小鼠(Gla KO/TgG3S)的肾脏、心脏、大脑和血浆中有更多的储存(图16A至图16D)。在雄性野生型小鼠的肾中，GL-3储存非常低，使得TgG3S小鼠中GL-3储存的轻微增加得以区分。GL-3在雄性Gla KO小鼠中的储存大于在TgG3S小鼠中的储存，并且与在Gla KO小鼠中观察到的水平相比，加重的Gla KO/TgG3S小鼠表现出显著增加的GL-3储存。GL-3在雌性小鼠肾组织中的储存表明GL-3储存从具有最低水平的野生型小鼠增加的明显趋势，接着是TgG3S和Gla KO小鼠，其中Gla KO/TgG3S小鼠具有最高的储存水平。Quantification of substrates (lyso-Gb₃ in plasma, GL-3 in tissues) by LC-MS/MS confirmed that these substrates had more storage in kidney, heart, brain and plasma of aggravated mice (Gla KO/TgG3S) compared with Gla KO mice (Figure 16A to Figure 16D). In the kidney of male wild-type mice, GL-3 storage was very low, making the slight increase of GL-3 storage in TgG3S mice distinguishable. The storage of GL-3 in male Gla KO mice was greater than that in TgG3S mice, and the aggravated Gla KO/TgG3S mice showed significantly increased GL-3 storage compared with the levels observed in Gla KO mice. The storage of GL-3 in female mouse kidney tissues showed a clear trend of GL-3 storage increasing from wild-type mice with the lowest levels, followed by TgG3S and Gla KO mice, with Gla KO/TgG3S mice having the highest storage levels.

在雄性动物的心脏组织中，在野生型和TgG3S小鼠中存在最低的GL-3储存。虽然GLA KO雄性小鼠在心脏组织中具有略微更多的GL-3储存，但加重的Gla KO/TgG3S小鼠具有显著增加的底物储存水平。在雌性小鼠中，在野生型小鼠的心脏组织中存在最低的GL-3储存，在TgG3S和Gla KO小鼠中具有略微增加的水平，并且在Gla KO/TgG3S小鼠中观察到显著增加的GL-3储存。In the cardiac tissue of male animals, there was the lowest GL-3 storage in wild-type and TgG3S mice. Although GLA KO male mice had slightly more GL-3 storage in cardiac tissue, aggravated Gla KO/TgG3S mice had significantly increased substrate storage levels. In female mice, there was the lowest GL-3 storage in cardiac tissue of wild-type mice, with slightly increased levels in TgG3S and Gla KO mice, and significantly increased GL-3 storage was observed in Gla KO/TgG3S mice.

在雄性和雌性小鼠的脑组织中，野生型、Gla KO和TgG3S小鼠的GL-3储存水平较低。在两种性别中，与所研究的其它三种模型相比，Gla KO/TgG3S小鼠在大脑中具有显著增加的GL-3储存。In brain tissue of male and female mice, GL-3 storage levels were lower in wild-type, Gla KO, and TgG3S mice. In both sexes, Gla KO/TgG3S mice had significantly increased GL-3 storage in the brain compared to the other three models studied.

在血浆中，lyso-Gb3储存水平在野生型和TgG3S小鼠中都是最低的。在雄性Gla KO和Gla KO/TgG3S小鼠中，这些水平增加到相似的程度。在雌性小鼠中，与野生型和TgG3S模型相比，Gla KO小鼠中的lyso-Gb3储存水平增加；然而，Gla KO和Gla KO/TgG3S小鼠之间的lyso-Gb3储存水平显著增加。In plasma, lyso-Gb3 storage levels were lowest in both wild-type and TgG3S mice. These levels increased to a similar extent in male Gla KO and Gla KO/TgG3S mice. In female mice, lyso-Gb3 storage levels were increased in Gla KO mice compared with wild-type and TgG3S models; however, lyso-Gb3 storage levels were significantly increased between Gla KO and Gla KO/TgG3S mice.

累积地，这项自然史研究证实过度表达人Gb3合酶的恶化的法布里病小鼠模型(Gla KO/TgG3S小鼠)在约18至25周龄(4.5至6月龄)开始出现疾病相关的异常。此外，GlaKO/TgG3S小鼠通常比非加重法布里病小鼠(Gla KO)表现出更严重的表型。具体地，与性别匹配的Gla KO小鼠相比，Gla KO/TgG3S小鼠表现出更严重的体重减轻(消瘦[雄性和雌性])和感觉缺陷(热板潜伏期增加[仅雄性])。Gla KO/TgG3S小鼠还表现出进行性肾损伤(血清BUN水平增加，尿渗透压降低[雄性和雌性])，这在Gla KO小鼠中并不明显，此外还表现出GL-3在肾脏、心脏、DRG、大脑和血浆中有更大的累积。加重的法布里病小鼠模型(Gla KO/TgG3S)还证实了在肾脏(单核变性间质性肾炎)和心脏(心肌细胞坏死和矿化)中从未观察到的一些继发性变性、坏死和矿化损伤，并且可能解释了更显著的表型。有趣的是，在肾小球中也观察到了储存材料，包括足细胞，类似于法布里病患者并且不同于Gla KO小鼠。足细胞(构成肾小球中的过滤屏障的细胞)中的储存是法布里病的病理生理学的关键，并且可能解释了小鼠加重模型和患者中增加的蛋白尿和降低的尿渗透压。Cumulatively, this natural history study demonstrated that an exacerbated Fabry disease mouse model (Gla KO/TgG3S mice) that overexpresses human Gb3 synthase begins to develop disease-related abnormalities at approximately 18 to 25 weeks of age (4.5 to 6 months of age). In addition, GlaKO/TgG3S mice generally exhibit a more severe phenotype than non-exacerbated Fabry disease mice (Gla KO). Specifically, Gla KO/TgG3S mice exhibited more severe weight loss (emaciation [males and females]) and sensory deficits (increased hot plate latency [males only]) compared to sex-matched Gla KO mice. Gla KO/TgG3S mice also exhibited progressive renal damage (increased serum BUN levels and decreased urine osmolality [males and females]) that was not evident in Gla KO mice, and also exhibited greater accumulation of GL-3 in the kidneys, heart, DRGs, brain, and plasma. The aggravated Fabry disease mouse model (Gla KO/TgG3S) also confirmed some secondary degeneration, necrosis and mineralization damage never observed in the kidney (mononuclear degeneration interstitial nephritis) and heart (cardiomyocyte necrosis and mineralization), and may explain the more significant phenotype. Interestingly, storage materials, including podocytes, were also observed in the glomeruli, similar to Fabry disease patients and different from Gla KO mice. Storage in podocytes (cells that constitute the filtration barrier in the glomerulus) is the key to the pathophysiology of Fabry disease and may explain the increased proteinuria and reduced urine osmotic pressure in the mouse aggravated model and patients.

GLA KO/TgG3S小鼠发展为进行性共济失调伴严重震颤和走动缺陷，促使其在35至40周龄左右实施安乐死，这与表现出正常寿命的Gla KO小鼠不同。这似乎可归因于CNS中显著的GL-3储存，包括在组织学上观察到浦肯野细胞(Purkinje cell)变性和丧失的小脑中。然而，浦肯野细胞变性和共济失调不是人类法布里病的特征。在加重的小鼠模型中，通过由普遍存在的启动子驱动的GL-3合酶的过表达实现Gla底物GL-3的人工过载。GL-3在CNS中的积累与在双突变体Gla KO/TgG3S中不存在GLA的情况下Gb3S的神经元过表达是连续的。因此，小鼠共济失调直接归因于CNS中广泛且显著的Gb3储存材料，且其可以通过将恢复GLA水平的基因治疗来减轻。由于这个原因，在加重的小鼠模型中对共济失调和存活的监测是小鼠的相关生物标志物，即使其不是翻译的生物标志物。GLA KO/TgG3S mice develop progressive ataxia with severe tremor and ambulation defects, prompting them to be euthanized at about 35 to 40 weeks of age, which is different from Gla KO mice that show normal life span. This seems to be attributable to significant GL-3 storage in the CNS, including in the cerebellum where Purkinje cell degeneration and loss are observed histologically. However, Purkinje cell degeneration and ataxia are not characteristics of human Fabry disease. In the aggravated mouse model, artificial overload of Gla substrate GL-3 is achieved by overexpression of GL-3 synthase driven by a ubiquitous promoter. The accumulation of GL-3 in the CNS is continuous with the neuronal overexpression of Gb3S in the absence of GLA in the double mutant Gla KO/TgG3S. Therefore, mouse ataxia is directly attributed to extensive and significant Gb3 storage materials in the CNS, and it can be alleviated by gene therapy that will restore GLA levels. For this reason, monitoring of ataxia and survival in mouse models of exacerbation is a relevant biomarker in mice, even though it is not a translational biomarker.

总之，本研究的结果支持使用加重的法布里病Gla KO/TgG3S小鼠模型作为具有以下疗效终点的疗效研究的测试系统：血浆中的lyso-Gb3储存、组织(肾脏、心脏、DRG、大脑)中的GL-3储存、组织病理学(肾脏、心脏、DRG、大脑)、热感觉功能(热板)、肾功能(BUN、尿渗透压)，共济失调和存活率。In conclusion, the results of this study support the use of the exacerbated Fabry disease Gla KO/TgG3S mouse model as a test system for efficacy studies with the following efficacy endpoints: lyso-Gb3 storage in plasma, GL-3 storage in tissues (kidney, heart, DRG, brain), histopathology (kidney, heart, DRG, brain), thermal sensory function (hot plate), renal function (BUN, urine osmolality), ataxia, and survival.

实例4：用于递送hGLA以进行基因治疗的rAAV载体的评估Example 4: Evaluation of rAAV Vectors for Delivery of hGLA for Gene Therapy

本研究的目的是确定用于基因治疗的最佳人α-半乳糖苷酶A(hGLA)氨基酸序列。测试了编码hGLA变体的构建体。为了公平比较，载体包括相同的衣壳和启动子，并且WPRE增强子也存在于所有表达盒中。The aim of this study was to determine the optimal human α-galactosidase A (hGLA) amino acid sequence for gene therapy. Constructs encoding hGLA variants were tested. For a fair comparison, the vectors included the same capsid and promoter, and the WPRE enhancer was also present in all expression cassettes.

对两至三月龄的法布里病小鼠(Gla KO)以下列剂量之一静脉内(IV)注射各种AAVhu68.hGLA载体：1x10¹¹GC(5x10¹²GC/kg-中剂量)或5x10¹¹GC(2.5x10¹³GC/kg-高剂量)。PBS处理的法布里病Gla KO和WT小鼠作为对照。在注射后(pi)1周和3周收集血液用于血清分离，并在pi 4周(尸检时间点)收集血液用于血浆分离。在pi 4周收集大脑、具有背根神经节(DRG)的脊髓、心脏、肾脏、肝脏、皮肤、小肠和大肠，其中一半处理用于组织学，另一半冷冻用于生物化学分析(通过定量质谱和GLA酶活性测量进行储存定量)。比较载体的主要疗效终点是靶器官中储存材料的定量。在Gla KO小鼠中，可以通过免疫组织化学(IHC)在锌-福尔马林石蜡包埋的组织切片上对储存材料酰基鞘鞍醇三己糖(GL-3)进行染色。肾小管上皮细胞中可见褐色沉积物，随着年龄的增长，储存逐渐恶化。储存还可以在H&E染色的DRG神经元中显现为放大的透明染色神经元(它们的透明颜色是由于细胞质中的糖脂储存材料)。法布里病的其它靶器官(如心脏、肠或脑脉管系统)在传统Gla KO小鼠模型中显示出低的和不一致的储存染色。收集并处理这些器官，但不允许对载体进行疗效评估。Two to three month old Fabry mice (Gla KO) were injected intravenously (IV) with various AAVhu68.hGLA vectors at one of the following doses: 1x10¹¹ GC (5x10¹² GC/kg - medium dose) or 5x10¹¹ GC (2.5x10¹³ GC/kg - high dose). PBS treated Fabry Gla KO and WT mice served as controls. Blood was collected at 1 and 3 weeks post-injection (pi) for serum separation and at 4 weeks pi (necropsy time point) for plasma separation. Brain, spinal cord with dorsal root ganglia (DRG), heart, kidney, liver, skin, small intestine and large intestine were collected at 4 weeks pi, half of which were processed for histology and the other half were frozen for biochemical analysis (depot quantification by quantitative mass spectrometry and GLA enzyme activity measurement). The primary efficacy endpoint for comparison of vectors was quantification of depot material in target organs. In Gla KO mice, the storage material ceramide (GL-3) can be stained by immunohistochemistry (IHC) on zinc-formalin paraffin embedded tissue sections. Brown deposits are visible in the renal tubular epithelial cells, and the storage gradually worsens with age. Storage can also be seen in H&E stained DRG neurons as enlarged transparent stained neurons (their transparent color is due to glycolipid storage materials in the cytoplasm). Other target organs of Fabry disease (such as the heart, intestines or brain vasculature) show low and inconsistent storage staining in traditional Gla KO mouse models. These organs are collected and processed, but do not allow for therapeutic evaluation of the vector.

Gla KO小鼠是广泛用于法布里病的模型(Ohshima T，Murray GJ，Swaim WD，Longenecker G，Quirk JM，Cardarelli CO，Sugimoto Y，Pastan I，Gottesman MM，BradyRO，Kulkarni AB：《α-半乳糖苷酶A缺陷型小鼠：法布里病模型(α-Galactosidase Adeficient mice：A model ofFabry disease)》《美国国家科学院院刊(Proc Natl AcadSci)》94：2540-2544，1997)。半合子雄性表现出异常的肾脏和肝脏形态，两者均具有酰基鞘鞍醇三己糖的累积。它们还表现出轻度心肌病和异常的心血管生理学。小尺寸、可再现的表型和有效的育种允许快速研究，其对于载体的临床前体内筛选是最佳的。Gla KO mice are widely used models of Fabry disease (Ohshima T, Murray GJ, Swaim WD, Longenecker G, Quirk JM, Cardarelli CO, Sugimoto Y, Pastan I, Gottesman MM, Brady RO, Kulkarni AB: "α-Galactosidase A deficient mice: A model of Fabry disease" "Proc Natl Acad Sci" 94: 2540-2544, 1997). Hemizygous males show abnormal kidney and liver morphology, both with accumulation of ceramide trihexose. They also show mild cardiomyopathy and abnormal cardiovascular physiology. Small size, reproducible phenotype and efficient breeding allow rapid research, which is optimal for preclinical in vivo screening of vectors.

比铰以下载体：Compared with the following carriers:

AAVhu68.CB7.hGLAnat.WPRE.rBGAAVhu68.CB7.hGLAnat.WPRE.rBG

AAVhu68.CB7.hGLAco.WPRE.rBGAAVhu68.CB7.hGLAco.WPRE.rBG

AAVhu68.CB7.hGLAco(M51C_G360C).rBGAAVhu68.CB7.hGLAco(M51C_G360C).rBG

IV途径的选择是由于容易进行、可再现性和稳健的肝和心脏转导，允许提取转基因GLA用于分析。它也是预期的临床施用途径。选择所选的剂量范围1x10¹¹GC至5x10¹¹GC(相当于大约5x10¹²GC/kg至2.5x10¹³GC/kg)以在最高剂量下实现肌肉、心脏和肝脏转导。预期最低剂量是次优的，因此可以更好地区分不同载体之间的疗效。The IV route was chosen due to ease of conduct, reproducibility and robust liver and heart transduction, allowing extraction of transgenic GLA for analysis. It is also the intended clinical route of administration. The selected dose range of 1x10¹¹ GC to 5x10¹¹ GC (equivalent to approximately 5x10¹² GC/kg to 2.5x10¹³ GC/kg) was chosen to achieve muscle, heart and liver transduction at the highest dose. The lowest dose is expected to be suboptimal, so the efficacy between different vectors can be better distinguished.

每组包括最少6只小鼠(雄性和雌性)以能够进行药理学读数的统计分析。A minimum of 6 mice (male and female) were included per group to enable statistical analysis of pharmacological readouts.

药理学读数包括生物化学测定(包括但不必限于GLA酶活性、酶总量的测定、与甘露糖-6-磷酸受体的结合、GL-3储存)和组织学终点(GL-3染色)。测定hGLA的抗体。Pharmacological readouts include biochemical assays (including but not necessarily limited to GLA enzyme activity, assays for total enzyme amount, binding to mannose-6-phosphate receptor, GL-3 storage) and histological endpoints (GL-3 staining). Assays for antibodies to hGLA.

表.组名称Table.Group Name

结果result

在注射后1周获得的血清样品中测量的GLA活性水平显示总体上，GLA活性水干是剂量依赖性的，其中在2.5x10¹³GC/kg剂量下在所有三种载体观察到更高的水平(图17)。与野生型和GLAKO对照相比，所有三种载体产生更高水平的GLA活性。在较高剂量组(2.5x10¹³GC/kg)中，与注射AAVhu68.hGLAco(M51C_G360C)的小鼠相比，施用AAVhu68.hGLAnat和AAVhu68.hGLAco的小鼠表现出更高水平的GLA活性。雄性小鼠比雌性小鼠表现出更高的酶活性，如预期的，这是由于与雌性小鼠相比，来自雄性的肝细胞中更有效的AAV转导和基因表达，这是非人灵长类和人中没有遇到的鼠特异性现象。在施用两种剂量AAVhu68.hGLAco(M51C_G360C)和低剂量(5.0x10¹²GC/kg)AAVhu68.hGLAnat和AAVhu68.hGLAco的雄性小鼠中，GLA活性相似。然而，在注射高剂量(2.5x10¹³GC/kg)AAVhu68.hGLAnat和AAVhu68.hGLAco的雄性中，GLA活性水平高得多。虽然雌性小鼠中的GLA活性水平低得多，但在雌性小鼠中也观察到雄性小鼠中观察到的三种载体中GLA活性水平的相同趋势。GLA activity levels measured in serum samples obtained 1 week after injection showed that overall, GLA activity levels were dose-dependent, with higher levels observed in all three vectors at a dose of 2.5x10¹³ GC/kg ( FIG. 17 ). All three vectors produced higher levels of GLA activity compared to wild-type and GLAKO controls. In the higher dose group (2.5x10¹³ GC/kg), mice administered AAVhu68.hGLAnat and AAVhu68.hGLAco showed higher levels of GLA activity compared to mice injected with AAVhu68.hGLAco (M51C_G360C). Male mice showed higher enzyme activity than female mice, as expected, due to more efficient AAV transduction and gene expression in hepatocytes from males compared to females, a mouse-specific phenomenon not encountered in non-human primates and humans. GLA activity was similar in male mice administered two doses of AAVhu68.hGLAco (M51C_G360C) and low doses (5.0x10¹² GC/kg) of AAVhu68.hGLAnat and AAVhu68.hGLAco. However, GLA activity levels were much higher in males injected with high doses (2.5x10¹³ GC/kg) of AAVhu68.hGLAnat and AAVhu68.hGLAco. Although GLA activity levels were much lower in female mice, the same trend in GLA activity levels observed in male mice for the three vectors was also observed in female mice.

在血清中测量的大部分酶活性来自在肝细胞中表达和分泌的蛋白质。为了研究用表达工程化候选物AAVhu68.hGLAco(M51C_G360C)的载体观察到的血清中更低酶活性的原因，我们在尸检时收集的肝样品中进行了载体基因组生物分布分析。对于所有三种载体，施用较高剂量(2.5x10¹³GC/kg)的小鼠表现出比用相应较低剂量的每种载体注射的小鼠更高的转导速率，并且3种不同载体在给定剂量水平下产生相似水平的载体基因组这表明编码工程化候选物的载体的酶活性降低可归因于转基因的表达降低(图18)。Most of the enzyme activity measured in serum comes from proteins expressed and secreted in hepatocytes. To investigate the cause of the lower enzyme activity in serum observed with vectors expressing the engineered candidate AAVhu68.hGLAco(M51C_G360C), we performed vector genome biodistribution analysis in liver samples collected at necropsy. For all three vectors, mice administered a higher dose (2.5x10¹³ GC/kg) exhibited higher transduction rates than mice injected with a correspondingly lower dose of each vector, and the 3 different vectors produced similar levels of vector genomes at a given dose level, suggesting that the reduced enzyme activity of the vector encoding the engineered candidate can be attributed to reduced expression of the transgene (Figure 18).

还在IV注射5.0x10¹²或2.5x10¹³GC/kg剂量的载体后第28天分析疾病相关器官中的组织酶活性水平。下图示出了心脏(图19)、肝脏(图20)、肾脏(图21)、大脑(图22)和小肠(图23)的酶活性结果。在施用高剂量(2.5x10¹³GC/kg)AAVhu68.hGLAnat和AAVhu68.hGLAco的小鼠中，心脏组织中测量的总GLA活性水平最高。在施用低剂量(5.0x10¹²GC/kg)的AAVhu68.hGLAnat和AAVhu68.hGLAco以及两种剂量的AAVhu68.hGLAco(M51C_G360C)的小鼠中观察到低得多的GLA活性水平。在雄性和雌性小鼠中均观察到相似的活性水平。Tissue enzyme activity levels in disease-related organs were also analyzed on day 28 after IV injection of 5.0x10¹² or 2.5x10¹³ GC/kg doses of vector. The following figure shows the results of enzyme activity in the heart (Figure 19), liver (Figure 20), kidney (Figure 21), brain (Figure 22) and small intestine (Figure 23). In mice administered with high doses (2.5x10¹³ GC/kg) of AAVhu68.hGLAnat and AAVhu68.hGLAco, the highest total GLA activity levels were measured in heart tissue. Much lower GLA activity levels were observed in mice administered with low doses (5.0x10¹² GC/kg) of AAVhu68.hGLAnat and AAVhu68.hGLAco and two doses of AAVhu68.hGLAco (M51C_G360C). Similar activity levels were observed in both male and female mice.

在肝脏中，总体GLA活性在施用高剂量(2.5x10¹³GC/kg)的所有三种AAVhu68.hGLA的小鼠中更高，并且在用AAVhu68.hGLAnat和AAVhu68.hGLAco处理的小鼠中最高。通常，与雌性小鼠相比，雄性小鼠的GLA活性略高。In the liver, total GLA activity was higher in mice administered a high dose (2.5 x¹⁰¹³ GC/kg) of all three AAVhu68.hGLA and was highest in mice treated with AAVhu68.hGLAnat and AAVhu68.hGLAco. In general, GLA activity was slightly higher in male mice compared to female mice.

虽然在肾组织中测量的总体GLA活性存在较小的剂量依赖性变化，但在施用高剂量(2.5x10¹³GC/kg)的所有三种AAVhu68.hGLA的小鼠中活性水平仍倾向于较高。在雄性和雌性小鼠之间观察到的GLA活性水平没有显著差异；然而，在雌性小鼠中测量的GLA活性水平有更多的可变性。Although there were minor dose-dependent changes in overall GLA activity measured in kidney tissue, activity levels still tended to be higher in mice administered the high dose (^2.5x1013 GC/kg) of all three AAVhu68.hGLA. No significant differences in GLA activity levels were observed between male and female mice; however, there was more variability in GLA activity levels measured in female mice.

在野生型小鼠的脑组织中观察到显著水干的GLA活性。同样，GLA活性水干在施用高剂量(2.5x10¹³GC/kg)的所有三种AAVhu68.hGLA的小鼠中更高，并且在用高剂量(2.5x10¹³GC/kg)的AAVhu68.hGLAnat和AAVhu68.hGLAco处理的小鼠中最高。在雄性和雌性小鼠中均观察到相似的活性水平。Significant amounts of GLA activity were observed in brain tissue of wild-type mice. Likewise, GLA activity levels were higher in mice administered a high dose (2.5x10¹³ GC/kg) of all three AAVhu68.hGLAs and were highest in mice treated with a high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAnat and AAVhu68.hGLAco. Similar activity levels were observed in both male and female mice.

用两种剂量的AAVhu68.hGLAco(M51C_G360C)和低剂量(5.0x10¹²GC/kg)的AAVhu68.hGLAnat和AAVhu68.hGLAco处理的小鼠的小肠中的GLA活性水平较低，并且在量级上相似。在小鼠高剂量(2.5x10¹³GC/kg)的AAVhu68.hGLAnat和AAVhu68.hGLAco中观察到最高水平的GLA活性。在雄性和雌性小鼠之间没有观察到显著的变化。The levels of GLA activity in the small intestine of mice treated with both doses of AAVhu68.hGLAco (M51C_G360C) and a low dose (5.0x10¹² GC/kg) of AAVhu68.hGLAnat and AAVhu68.hGLAco were low and similar in magnitude. The highest levels of GLA activity were observed in mice treated with a high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAnat and AAVhu68.hGLAco. No significant changes were observed between male and female mice.

总之，与上述血清结果一致，GLA酶活性的组织水平是剂量依赖性的，在编码未修饰的天然蛋白质(工程化序列或未工程化序列)的两个候选物之间是相当的，并且在编码工程化蛋白质hGLAco(M51C_G360C)的候选物中显著较低。在肝脏以外的器官中未观察到性别影响。In summary, consistent with the above serum results, tissue levels of GLA enzyme activity were dose-dependent, comparable between the two candidates encoding the unmodified native protein (engineered or unengineered sequence), and significantly lower in the candidate encoding the engineered protein hGLAco (M51C_G360C). No gender effects were observed in organs other than the liver.

为了进一步评估这三种载体的药理学，我们在AAV施用后28天通过LC-MS/MS测量了来自GLA KO小鼠的血浆中的储存材料lyso-Gb3和组织中的GL-3的量，并将这些水平与在PBS处理的GLA KO和野生型对照小鼠中测量的那些水平进行了比较。Lyso-Gb3和GL-3储存减少与酶活性水平一致；编码GLA的两种载体(AAVhu68.hGLAnat和AAVhu68.hGLAco)导致血浆、肾和心脏样品中高剂量(2.5x10¹³GC/kg)的完全储存消除(图24)。然而，编码hGLAco(M51C_G360C)的载体仅部分降低了血浆中的lyso-Gb3储存水平以及肾和心脏组织中的GL-3储存水平。To further evaluate the pharmacology of these three vectors, we measured the amount of storage material lyso-Gb3 in plasma and GL-3 in tissues from GLA KO mice by LC-MS/MS 28 days after AAV administration, and compared these levels with those measured in GLA KO and wild-type control mice treated with PBS. Lyso-Gb3 and GL-3 storage reductions were consistent with enzyme activity levels; two vectors encoding GLA (AAVhu68.hGLAnat and AAVhu68.hGLAco) resulted in complete storage elimination of high doses (2.5x10¹³ GC/kg) in plasma, kidney and heart samples (Figure 24). However, the vector encoding hGLAco (M51C_G360C) only partially reduced the lyso-Gb3 storage levels in plasma and the GL-3 storage levels in kidney and heart tissues.

实例5：用于递送hGLA以进行基因治疗的rAAV载体的评估Example 5: Evaluation of rAAV vectors for delivering hGLA for gene therapy

本研究的目的是评估多达三种不同剂量(2.5x10¹²GC/kg，5x10¹²GC/kg，2.5x10¹³GC/kg)的三种载体，以确定IV施用后在Gla KO小鼠中的疗效。所有评估的载体具有相同的衣壳、启动子和polyA信号，但包括不同版本的人GLA转基因。所评估的三种转基因是hGALco(与实例4中相同)、hGLAco(M51C_G360C)(与实例4中相同)和hGLA-D233C-1359Cco。hGLAco(M51C_G360C)转基因编码具有两个点突变的工程化GLA蛋白，点突变引入二硫键以使酶稳定在其活性二聚体形式。hGLAco(D233C_I359C)转基因编码具有两个点突变的工程化GLA蛋白的第二版本，点突变引入二硫键以使酶稳定在其活性二聚体形式。The purpose of this study is to evaluate three vectors at up to three different doses (2.5x10¹² GC/kg, 5x10¹² GC/kg, 2.5x10¹³ GC/kg) to determine the efficacy in Gla KO mice after IV administration. All evaluated vectors have the same capsid, promoter and polyA signal, but include different versions of human GLA transgenes. The three transgenes evaluated are hGALco (same as in Example 4), hGLAco (M51C_G360C) (same as in Example 4) and hGLA-D233C-1359Cco. The hGLAco (M51C_G360C) transgene encodes an engineered GLA protein with two point mutations that introduce disulfide bonds to stabilize the enzyme in its active dimer form. The hGLAco (D233C_I359C) transgene encodes a second version of an engineered GLA protein with two point mutations that introduce disulfide bonds to stabilize the enzyme in its active dimer form.

成年小鼠(3.5至4.5月龄)接受单次IV施用3种候选载体(AAVhu68.hGLAco、AAVhu68.hGLAco(M51C_G360C)或AAVhu68.hGLAco(D233C_I359C)中的1种，低剂量为2.5x10¹²GC/kg，中剂量为5.0x10¹²GC/kg，或高剂量为2.5x10¹³GC/kg(仅AAVhu68.hGLAco(D233C_I359C))。媒介物(PBS)处理的WT和Gla KO小鼠作为对照。Adult mice (3.5 to 4.5 months old) received a single IV administration of 1 of 3 candidate vectors (AAVhu68.hGLAco, AAVhu68.hGLAco(M51C_G360C), or AAVhu68.hGLAco(D233C_I359C) at a low dose of^2.5x1012 GC/kg, a mid dose of^5.0x1012 GC/kg, or a high dose of^2.5x1013 GC/kg (AAVhu68.hGLAco(D233C_I359C) only). Vehicle (PBS) treated WT and Gla KO mice served as controls.

每天监测动物的活力。在第7天收集血清以评估转基因产物表达(GLA酶活性)。在第28天，进行尸检，收集具有DRG的心脏、肾、肝和脊髓，并处理用于组织学、GL-3定量和GLA酶活性的评估。还收集血浆用于lyso-Gb₃定量和评估GLA酶活性。The vitality of animals was monitored every day. Serum was collected at the 7th day to assess transgenic product expression (GLA enzymatic activity). At the 28th day, autopsy was performed, and the heart, kidney, liver and spinal cord with DRG were collected, and the assessment of histology, GL-3 quantification and GLA enzymatic activity was processed. Blood plasma was also collected for lyso-Gb₃ quantification and assessment GLA enzymatic activity.

静脉内施用对所有评估的载体都具有良好的耐受性。所有动物均存活至预定的尸检时间点。Intravenous administration was well tolerated for all vehicles evaluated. All animals survived until the scheduled necropsy time point.

在雄性和雌性Gla KO鼠的汇总数据中，在施用高剂量(2.5x10¹³GC/kg)AAVhu68.hGLAco(D233C_I359C)的Gla KO小鼠中，血清转基因产物表达(GLA酶活性)最高。GLA酶活性水平在其余处理组中的Gla KO小鼠中相似，尽管在施用AAVhu68.hGLAco或AAVhu68.hGLAco(D233C_I359C)的Gla KO小鼠中存在轻微的剂量依赖性应答。雄性Gla KO小鼠表现出比雌性更高的GLA酶活性。在施用高剂量(2.5x10¹³GC/kg)AAVhu68.hGLAco(D233C_I359C)的雄性Gla KO小鼠中GLA酶活性最高，并且在施用AAVhu68.hGLAco或AAVhu68.hGLAco(D233C_I359C)的雄性Gla KO小鼠中存在一定剂量依赖性的GLA酶活性。雌性Gla KO小鼠中的GLA酶活性非常低，在施用高剂量(2.5x10¹³GC/kg)AAVhu68.hGLAco(D233C_I359C)的小鼠中观察到最高水平(图25)。In the pooled data of male and female Gla KO mice, serum transgene product expression (GLA enzyme activity) was highest in Gla KO mice administered a high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C). GLA enzyme activity levels were similar in Gla KO mice in the remaining treatment groups, although there was a slight dose-dependent response in Gla KO mice administered AAVhu68.hGLAco or AAVhu68.hGLAco(D233C_I359C). Male Gla KO mice exhibited higher GLA enzyme activity than females. The highest GLA enzyme activity was observed in male Gla KO mice administered with a high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C), and there was a dose-dependent GLA enzyme activity in male Gla KO mice administered with AAVhu68.hGLAco or AAVhu68.hGLAco(D233C_I359C). The GLA enzyme activity in female Gla KO mice was very low, with the highest level observed in mice administered with a high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C) ( FIG. 25 ).

在施用后28天收集的血浆中测量的转基因产物表达(GLA酶活性)的汇总数据揭示了所研究的所有3种AAV载体的明显剂量依赖性效应，其中在施用中剂量(5.0x10¹²GC/kg)AAVhu68.hGLAco和高剂量(2.5x10¹³GC/kg)AAVhu68.hGLAco(D233C_I359C)的Gla KO小鼠中观察到最高水平的GLA酶活性。GLA酶活性在雄性Gla KO小鼠中比在雌性Gla KO小鼠中高得多。在雄性Gla KO小鼠中观察到AAVhu68.hGLA对所有供试品的GLA活性的剂量依赖性作用，其中中剂量(5.0x10¹²GC/kg)的AAVhu68.hGLAco和高剂量(2.5x10¹³GC/kg)的AAVhu68.hGLAco(D233C_I359C)产生最高的酶活性。在雌性Gla KO小鼠中GLA活性水平普遍较低，高剂量(2.5x10¹³GC/kg)的AAVhu68.hGLAco(D233C_I359C)提供最高水平的活性(图26)。The pooled data of transgene product expression (GLA enzyme activity) measured in plasma collected 28 days after administration revealed a clear dose-dependent effect for all three AAV vectors studied, with the highest levels of GLA enzyme activity observed in Gla KO mice administered with a medium dose (5.0x10¹² GC/kg) of AAVhu68.hGLAco and a high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C). GLA enzyme activity was much higher in male Gla KO mice than in female Gla KO mice. A dose-dependent effect of AAVhu68.hGLA on GLA activity of all tested articles was observed in male Gla KO mice, with the medium dose (5.0x10¹² GC/kg) of AAVhu68.hGLAco and the high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C) producing the highest enzyme activity. GLA activity levels were generally lower in female Gla KO mice, with the high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C) providing the highest level of activity ( FIG. 26 ).

图27、图28和图29分别示出了心脏、肝脏和肾组织中的GLA酶活性。Figure 27, Figure 28 and Figure 29 show GLA enzyme activity in heart, liver and kidney tissues, respectively.

心脏组织中GLA酶活性的汇总数据显示所研究的所有3种AAV载体都具有明显的剂量依赖性效应，其中在施用中剂量(5.0x10¹²GC/kg)AAVhu68.hGLAco和高剂量(2.5x10¹³GC/kg)AAVhu68.hGLAco(D233C_I359C)的Gla KO小鼠中观察到最高水平的GLA酶活性。在雄性和雌性Gla KO小鼠中观察到相似水平的GLA酶活性。The summary data of GLA enzyme activity in cardiac tissue showed a clear dose-dependent effect for all three AAV vectors studied, with the highest levels of GLA enzyme activity observed in Gla KO mice administered with a medium dose (5.0x10¹² GC/kg) of AAVhu68.hGLAco and a high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C). Similar levels of GLA enzyme activity were observed in male and female Gla KO mice.

肝脏样品中GLA酶活性的综合数据揭示了施用两剂量AAVhu68.hGLAco和高剂量(2.5x10¹³GC/kg)AAVhu68.hGLAco(D233C_I359C)的Gla KO小鼠中的酶活性水平最高。这些观察结果反映在雄性Gla KO小鼠的结果中。雌性Gla KO小鼠具有比雄性Gla KO小鼠显著更低水平的GLA酶活性，并且在三种AAV载体和剂量之间表现出较小的活性差异。The combined data of GLA enzyme activity in liver samples revealed the highest levels of enzyme activity in Gla KO mice administered two doses of AAVhu68.hGLAco and a high dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C). These observations were reflected in the results for male Gla KO mice. Female Gla KO mice had significantly lower levels of GLA enzyme activity than male Gla KO mice and showed less activity difference between the three AAV vectors and doses.

对于雄性和雌性Gla KO小鼠，在肾脏样品中测量的GLA酶活性显示对于所研究的所有3种AAVhu68.hGLA载体的明显的剂量依赖性效应，在施用中剂量(5.0x10¹²GC/kg)的AAVhu68.hGLAco和AAVhu68.hGLAco(M51C_G360C)和高剂量(2.5x10¹³GC/kg)的AAVhu68.hGLAco(D233C_I359C)的小鼠中具有最高水平的活性。当通过性别分析GLA酶活性时，这些趋势得到了反映，这也揭示了雄性和雌性Gla KO小鼠中相似的GLA酶活性水平。For both male and female Gla KO mice, GLA enzyme activity measured in kidney samples showed a clear dose-dependent effect for all three AAVhu68.hGLA vectors studied, with the highest levels of activity in mice administered the mid-dose (5.0x10¹² GC/kg) of AAVhu68.hGLAco and AAVhu68.hGLAco(M51C_G360C) and the high-dose (2.5x10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C). These trends were mirrored when GLA enzyme activity was analyzed by sex, which also revealed similar levels of GLA enzyme activity in male and female Gla KO mice.

评估来自经处理的Gla KO小鼠的血浆以评估载体在减少lyso-Gb₃储存方面的疗效(图30)。数据显示用中剂量(5.0x10¹²GC/kg)AAVhu68.hGLAco和中剂量和高剂量(分别为5.0x10¹²GC/kg和2.5x10¹³GC/kg)AAVhu68.hGLAco(D233C_I359C)处理Gla KO小鼠完全清除了血浆中的lyso-Gb₃储存。这些结果在雄性和雌性Gla KO小鼠中都是一致的。Plasma from treated Gla KO mice was evaluated to assess the efficacy of the vector in reducing lyso-Gb₃ stores (Figure 30). The data showed that treatment of Gla KO mice with a medium dose (5.0x10¹² GC/kg) of AAVhu68.hGLAco and a medium and high dose (5.0x10¹² GC/kg and 2.5x10¹³ GC/kg, respectively) of AAVhu68.hGLAco(D233C_I359C) completely eliminated lyso-Gb₃ stores in plasma. These results were consistent in both male and female Gla KO mice.

来自肾组织样品的免疫组织化学数据揭示，虽然在施用所有三种载体的最高剂量下观察到GL-3储存的一些减少，但在用高剂量(2.5x10¹³GC/kg)AAVhu68.hGLAco(D233C_I359C)处理的Gla KO小鼠中观察到GL-3储存的最明显减少(图31A)。与先前的结果一致，肾脏IHC染色的GL-3储存的定量揭示与媒介物处理的Gla KO对照相比，用所有3个剂量的AAVhu68.hGLAco(D233C_I359C)处理的Gla KO小鼠在肾小管中具有显著更少的GL-3储存。用AAVhu68.hGLAco或其它工程化变体AAVhu68.hGLAco(M51C_G360C)处理的小鼠均没有显著的储存减少。观察到GL-3储存的这种减少是剂量依赖性的，在施用最高剂量(2.5x10¹³GC/kg)的AAVhu68.hGLAco(D233C_I359C)的Gla KO小鼠中具有最大效果(图31B)。Immunohistochemistry data from kidney tissue samples revealed that, while some reduction in GL-3 storage was observed at the highest dose of all three vectors, the most pronounced reduction in GL-3 storage was observed in Gla KO mice treated with the high dose (2.5 x 10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C) ( FIG. 31A ). Consistent with previous results, quantification of GL-3 storage by renal IHC staining revealed that Gla KO mice treated with all 3 doses of AAVhu68.hGLAco(D233C_I359C) had significantly less GL-3 storage in the renal tubules compared to vehicle-treated Gla KO controls. None of the mice treated with AAVhu68.hGLAco or other engineered variants, AAVhu68.hGLAco(M51C_G360C), had a significant reduction in storage. This reduction in GL-3 stores was observed to be dose-dependent, with the greatest effect in Gla KO mice administered the highest dose (2.5×10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C) ( FIG. 31B ).

来自DRG纵向切片的免疫组织化学数据显示，与用媒介物、AAVhu68.hGLAco或AAVhu68.hGLAco(M51C_G360C)处理的Gla KO小鼠相比，用AAVhu68.hGLAco(D233C_I359C)处理的Gla KO小鼠具有最少的GL-3储存(图32A)。对这些IHC数据的定量显示，与媒介物处理的Gla KO小鼠相比，在用所有三个剂量的AAVhu68.hGLAco(D233C_1359C)处理的Gla KO小鼠中DRG神经元GL-3储存显著减少。观察到这种应答是剂量依赖性的，在施用最高剂量(2.5x10¹³GC/kg)的AAVhu68.hGLAco(D233C_I359C)的小鼠中具有最大效果。同样，其它候选物AAVhu68.hGLAco和其它工程化变体AAVhu68.hGLAco(M51C_G360C)不导致DRG中显著的GL-3储存减少(图32B)。Immunohistochemistry data from DRG longitudinal sections showed that Gla KO mice treated with AAVhu68.hGLAco(D233C_I359C) had minimal GL-3 storage compared to Gla KO mice treated with vehicle, AAVhu68.hGLAco, or AAVhu68.hGLAco(M51C_G360C) ( FIG. 32A ). Quantification of these IHC data showed that DRG neuronal GL-3 storage was significantly reduced in Gla KO mice treated with all three doses of AAVhu68.hGLAco(D233C_1359C) compared to vehicle treated Gla KO mice. This response was observed to be dose dependent, with the greatest effect in mice administered the highest dose (2.5×10¹³ GC/kg) of AAVhu68.hGLAco(D233C_I359C). Likewise, other candidates AAVhu68.hGLAco and other engineered variants AAVhu68.hGLAco(M51C_G360C) did not result in significant reduction of GL-3 storage in DRGs ( FIG. 32B ).

累积地，AAVhu68.hGLAco(D233C_I359C)施用至Gla KO小鼠导致显著的转基因产物表达(GLA酶活性)，与所评估的其它载体相比具有最高的血浆和组织体内疗效。与媒介物处理的Gla KO小鼠相比，AAVhu68.hGLAco(D233C_I359C)处理的Gla KO小鼠表现出肾脏和DRG GL-3储存的显著剂量依赖性减少，而在相同剂量水平下施用非工程化AAVhu68.hGLAco载体不显著减少GL-3储存。Cumulatively, administration of AAVhu68.hGLAco(D233C_I359C) to Gla KO mice resulted in significant transgene product expression (GLA enzyme activity) with the highest plasma and tissue in vivo efficacy compared to other vectors evaluated. Gla KO mice treated with AAVhu68.hGLAco(D233C_I359C) exhibited a significant dose-dependent reduction in renal and DRG GL-3 storage compared to vehicle-treated Gla KO mice, while administration of non-engineered AAVhu68.hGLAco vectors at the same dose level did not significantly reduce GL-3 storage.

实例6：非人灵长类动物中AAVhu68.hGLAco(D233C_I359C)的评估Example 6: Evaluation of AAVhu68.hGLAco(D233C_I359C) in non-human primates

本研究旨在评估向食蟹猴静脉内施用AAVhu68.hGLAco(D233C_I359C)的初步药理学和安全性。The aim of this study was to evaluate the preliminary pharmacology and safety of intravenous administration of AAVhu68.hGLAco(D233C_I359C) to cynomolgus monkeys.

成年NHP(N＝4)接受单次IV剂量的AAVhu68.hGLAco(D233C_I359C)，剂量为2.5x10¹³GC/kg。活体评估包括每日进行的临床观察、体重、血液临床病理学(CBC、凝血面板、血清化学[包括心脏生物标志物肌钙蛋白I])和心脏功能评估(EKG和超声心动图)。在载体施用后的第60±3天，对所有动物进行尸检。尸检时，收集组织进行组织病理学检查。收集靶组织用于载体生物分布分析和转基因产物表达定位(人GLA ISH[mRNA]和人GLAIHC[蛋白质])的综合组织学评估。还收集PBMC、脾细胞和肝淋巴细胞以测量对衣壳和转基因产物(IFN-γELIspot)的T细胞应答。研究设计如下表所示。Adult NHP (N=4) received a single IV dose of AAVhu68.hGLAco (D233C_I359C) at a dose of 2.5x10¹³ GC/kg. In vivo assessments included daily clinical observations, body weight, blood clinical pathology (CBC, coagulation panel, serum chemistry [including cardiac biomarker troponin I]), and cardiac function assessment (EKG and echocardiogram). All animals were necropsied on day 60±3 after vector administration. At necropsy, tissues were collected for histopathological examination. Target tissues were collected for comprehensive histological assessment of vector biodistribution analysis and transgene product expression localization (human GLA ISH [mRNA] and human GLAIHC [protein]). PBMCs, splenocytes, and liver lymphocytes were also collected to measure T cell responses to capsid and transgene products (IFN-γ ELIspot). The study design is shown in the table below.

^aBL在给药前长达21天。^a BL up to 21 days prior to dosing.

^b血清化学分析包括肌钙蛋白I(心脏生物标志物)。^b Serum chemistry analysis included troponin I (cardiac biomarker).

^c收集样品并储存以备将来分析。^c Collect samples and store for future analysis.

^d收集组织用于组织病理学、载体生物分布分析和转基因产物表达(人GLA ISH染色[mRNA]和人GLA IHC染色[蛋白质])的组织学评估。收集肝和脾淋巴细胞用于测量对衣壳或转基因产物的T细胞应答(IFN-γ ELISpot)。^d Tissues were collected for histopathology, vector biodistribution analysis, and histological assessment of transgene product expression (human GLA ISH staining [mRNA] and human GLA IHC staining [protein]). Liver and spleen lymphocytes were collected for measurement of T cell responses to capsid or transgene product (IFN-γ ELISpot).

缩写：ADA，抗药物抗体；BL，基线；ELISpot，酶联免疫斑点技术；GLA，α-半乳糖苷酶；IFN-γ，干扰素γ；IHC，免疫组织化学；IN，鼻内；ISH，原位杂交；mRNA，信使核糖核酸；MS，质谱；Nab，中和抗体；PBMC，外周血单核细胞Abbreviations: ADA, antidrug antibody; BL, baseline; ELISpot, enzyme-linked immunospot; GLA, α-galactosidase; IFN-γ, interferon gamma; IHC, immunohistochemistry; IN, intranasal; ISH, in situ hybridization; mRNA, messenger RNA; MS, mass spectrometry; Nab, neutralizing antibody; PBMC, peripheral blood mononuclear cell

基线样品收集：Baseline sample collection:

在给药供试品或对照品(基线)之前长达21天以及在下表中所示的时间点从所有动物收集基线血液样品，包括全血计数(CBC)、凝血、心脏生物标志物、血清化学和PBMC/ELISPOT。在样品收集之前，从每只动物获得生命体征(即，温度、心率、呼吸)。Baseline blood samples including complete blood count (CBC), coagulation, cardiac biomarkers, serum chemistry and PBMC/ELISPOT were collected from all animals up to 21 days before dosing with the test or control articles (baseline) and at the time points shown in the table below. Prior to sample collection, vital signs (i.e., temperature, heart rate, respiration) were obtained from each animal.

a)衣壳中和抗体(血清)：在基线和第60天(尸检)时收集用于检测AAVhu68 Nab存在的血液(至多2mL)。通过红顶试管(有或没有血清分离器)收集血液，使其凝结，并离心。a) Capsid Neutralizing Antibodies (Serum): Blood (up to 2 mL) for the presence of AAVhu68 Nab was collected at baseline and day 60 (necropsy). Blood was collected via red-top tubes (with or without a serum separator), allowed to clot, and centrifuged.

b)心脏生物标志物(血清)：在基线、第3天、第7天、第14天、第28天和第60天(尸检)收集用于检测心脏毒性标志物(肌钙蛋白I)存在的血液(至多2mL)。通过红顶试管(有或没有血清分离器)收集血液，使其凝结，并离心。分离出血清。b) Cardiac Biomarkers (Serum): Blood (up to 2 mL) for the presence of a cardiotoxicity marker (troponin I) was collected at baseline, day 3, day 7, day 14, day 28, and day 60 (necropsy). Blood was collected in red-top tubes (with or without a serum separator), allowed to clot, and centrifuged. Serum was separated.

c)转基因表达、抗体、补体因子或细胞因子(血浆)：在第0天、第3天、第7天、第14天、第28天和第60天(尸检)收集用于检测hGLA、抗hGLAAb和/或补体活化或细胞因子(在毒性的情况下)存在的血液(至少3mL)。将血液收集在标记的淡紫色盖子(EDTAK2)中并在收集后30分钟内在约+4℃离心机中以约2700RPM(1300±100x g)离心15分钟。c) Transgene expression, antibodies, complement factors or cytokines (plasma): Blood (at least 3 mL) for the presence of hGLA, anti-hGLAAb and/or complement activation or cytokines (in case of toxicity) was collected on days 0, 3, 7, 14, 28 and 60 (necropsy). Blood was collected in labeled lavender caps (EDTA K2) and centrifuged at approximately 2700 RPM (1300 ± 100 x g) in a centrifuge at approximately +4°C for 15 minutes within 30 minutes of collection.

d)PBMC/ELISPOT：将血液(5至10mL)收集到肝素钠(绿顶试管)中并分离PBMC。在基线和尸检时收集样品。评估对衣壳和/或转基因的T细胞应答。d) PBMC/ELISPOT: Collect blood (5 to 10 mL) into sodium heparin (green top tubes) and isolate PBMCs. Collect samples at baseline and necropsy. Evaluate T cell responses to capsid and/or transgene.

e)血液学(细胞计数和分类)：收集血液(至多2mL)以获得具有差异和血小板计数的全血计数。在研究设计中指定的特定时间点分析以下参数：e) Hematology (cell count and differential): Blood (up to 2 mL) was collected to obtain a complete blood count with differential and platelet count. The following parameters were analyzed at specific time points specified in the study design:

红细胞计数Red blood cell count

血红蛋白Hemoglobin

血细胞比容Hematocrit

平均红细胞体积(MCV)Mean Corpuscular Volume (MCV)

平均红细胞血红蛋白(MCH)Mean Corpuscular Hemoglobin (MCH)

平均红细胞血红蛋白浓度(MCHC)Mean Corpuscular Hemoglobin Concentration (MCHC)

血小板计数Platelet count

白细胞计数White blood cell count

白细胞分类WBC Classification

红细胞形态(病理学家审查)Red blood cell morphology (reviewed by pathologist)

网织红细胞计数Reticulocyte count

f)临床化学：将用于临床化学研究的血液(至多2.0mL)收集在标记的红顶(血清)试管中，使其凝结至多15分钟，并离心。分离血清并置于标记的微量离心管中。在研究设计中指定的特定时间点分析以下参数：f) Clinical Chemistry: Blood (up to 2.0 mL) for clinical chemistry studies was collected in labeled red-top (serum) tubes, allowed to clot for up to 15 minutes, and centrifuged. Serum was separated and placed in labeled microcentrifuge tubes. The following parameters were analyzed at specific time points specified in the study design:

碱性磷酸酶Alkaline phosphatase

溶血标志物：胆红素(直接，间接)Hemolysis markers: bilirubin (direct, indirect)

肌酐Creatinine

γ-谷氨酰转肽酶γ-Glutamyltransferase

葡萄糖glucose

血清丙氨酸转氨酶Serum alanine aminotransferase

血清天冬氨酸转氨酶Serum aspartate aminotransferase

白蛋白albumin

白蛋白/球蛋白比(计算值)Albumin/globulin ratio (calculated value)

血尿素氮Blood urea nitrogen

g)凝血：将用于凝血面板的血液(2.0mL)收集在标记的蓝顶(柠檬酸盐)试管中。在研究设计中指定的特定时间点分析以下参数：g) Coagulation: Blood (2.0 mL) for the coagulation panel was collected in labeled blue-top (citrate) tubes. The following parameters were analyzed at specific time points specified in the study design:

PTTPTT

PTPT

纤维蛋白原Fibrinogen

D-二聚体D-Dimer

纤维蛋白降解产物Fibrin degradation products

心脏监测Heart monitoring

研究涉及载体给药前的基线超声心动图和研究终止时的额外回声。评估的最小参数包括舒张/收缩容积、每搏输出量、心输出量、缩短分数、隔膜厚度、射血时间。还评估了心尖两腔或四腔、右胞骨旁长轴四腔视图、右胸骨旁短轴视图的射血分数，当与心率组合时，得出左心室射血分数、舒张末期容积、收缩末期容积、每搏输出量和心输出量。The study involved a baseline echocardiogram before vehicle administration and an additional echo at study termination. Minimal parameters assessed included diastolic/systolic volumes, stroke volume, cardiac output, fractional shortening, septal thickness, and ejection time. Ejection fraction was also assessed in the apical two-chamber or four-chamber, right parasternal long-axis four-chamber view, and right parasternal short-axis view, which, when combined with heart rate, yielded left ventricular ejection fraction, end-diastolic volume, end-systolic volume, stroke volume, and cardiac output.

结果：result:

基于临床观察，静脉内施用是良好耐受的，并且所有动物存活至预定的尸检时间点。在基线、第14天、第28天和第60天时，所有动物的肌钙蛋白I水平(可指示心肌细胞损伤)均低于0.200μg/L至180μg/L的可报告范围，ECG和心电描记术上未发现异常。Based on clinical observations, intravenous administration was well tolerated and all animals survived to the scheduled necropsy time point. At baseline, day 14, day 28, and day 60, all animals had troponin I levels (indicative of myocardial cell damage) below the reportable range of 0.200 μg/L to 180 μg/L, and no abnormalities were found on ECG and electrocardiography.

在血液临床病理学上，发现包括在第3天所有动物中AST和ALT水平的短暂升高，并且在第7天至第14天在没有干预的情况下得到消退(图36A和图36B)。In blood clinical pathology, findings included transient elevations in AST and ALT levels in all animals on day 3, which resolved without intervention between days 7 and 14 ( FIGS. 36A and 36B ).

在整个研究中，所有动物的总胆红素(TBil)水平、血小板计数和白细胞(WBC)计数保持在正常限度内(图37A、图37B和图37C)。Total bilirubin (TBil) levels, platelet counts, and white blood cell (WBC) counts remained within normal limits in all animals throughout the study (Figure 37A, Figure 37B, and Figure 37C).

在整个研究中从动物收集的凝血数据揭示在第3天几只动物中凝血酶原时间(PT)、活化的APTT和D-二聚体水平的短暂升高(图38A，图38B和图38C)。这些短暂升高在没有干预的情况下得到消退。Coagulation data collected from animals throughout the study revealed transient increases in prothrombin time (PT), activated APTT, and D-dimer levels in several animals on day 3 (Figure 38A, Figure 38B, and Figure 38C). These transient increases resolved without intervention.

对于所评估的任何肽库，通过IFN-γELISpot在来自施用单一IV剂量的AAVhu68.hGLAco(D233C_1359C)的动物的来自脾、心脏淋巴结或肝的PBMC或淋巴细胞中未观察到针对人GLA转基因产物或AAVhu68衣壳的T细胞应答。No T cell responses to the human GLA transgene product or AAVhu68 capsid were observed by IFN-γ ELISpot in PBMCs or lymphocytes from the spleen, cardiac lymph nodes, or liver from animals administered a single IV dose of AAVhu68.hGLAco(D233C_1359C) for any of the peptide pools evaluated.

尸检时收集的组织的组织病理学检查揭示在一些器官中存在一些轻微(1级)炎性细胞浸润，其发生率和严重程度与历史对照和关于背景发现的已发表文献中的典型背景发现相似。DRG和TRG神经元变性和脊髓背轴突病不存在或极少。Histopathological examination of tissues collected at necropsy revealed some mild (grade 1) inflammatory cell infiltration in some organs, similar in incidence and severity to typical background findings in historical controls and published literature on background findings. DRG and TRG neuronal degeneration and dorsal spinal cord axonopathy were absent or minimal.

表：单次静脉内施用AAVhu68.hGLAco(D233C_I359C)(2.5x10¹³GC/kg)后60天从成年NHP收集的组织的半定量组织病理学评分评估Table: Semi-quantitative histopathology scoring assessment of tissues collected from adult NHPs 60 days after a single intravenous administration of AAVhu68.hGLAco(D233C_I359C) (^2.5x1013 GC/kg)

^a数字表示发现的半定量分级。0＝在正常限度内，1＝最小严重度。通过将观察到的1级结果的数量相加并除以对该组织评估的总切片，确定脊髓和DRG小于1级(计算在括号中提供)。^a Numbers indicate semiquantitative grading of findings. 0 = within normal limits, 1 = minimal severity. Spinal cord and DRG were determined to be less than grade 1 by adding the number of grade 1 findings observed and dividing by the total sections evaluated for that tissue (calculation is provided in parentheses).

抗AAVhu68衣壳的中和抗体和非中和结合抗体(BAb)(即，免疫球蛋白G[IgG]和免疫球蛋白M[IgM])在基线时在任何NHP中均未达到可检测水平，这与本研究的NAb阴性动物的筛选一致(图39)。正如所预期的，到第60天，所有动物都具有可检测水平的抗AAVhu68衣壳的NAb和IgG BAb，而抗AAVhu68衣壳的IgM BAb保持低于可检测限度。Neutralizing antibodies and non-neutralizing binding antibodies (BAbs) (i.e., immunoglobulin G [IgG] and immunoglobulin M [IgM]) against AAVhu68 capsid did not reach detectable levels in any NHP at baseline, consistent with the screening of NAb-negative animals in this study (Figure 39). As expected, by day 60, all animals had detectable levels of NAbs and IgG BAbs against AAVhu68 capsid, while IgM BAbs against AAVhu68 capsid remained below the detectable limit.

在血浆中，IV施用AAVhu68.hGLAco(D233C_I359C)导致显著水平的转基因产物表达(GLA酶活性)。平均GLA酶活性从施用后第0天至第14天增加大约50倍(图40)。GLA酶活性水平在第14天达到峰值，然后在第60天下降。在评估的最后时间点(第60天)，GLA酶活性比在第0天观察到的基线水平高大约2倍。在第14天之后转基因产物表达的这种下降是意料之中的。类似于先前对AAV递送人转基因产物的NHP研究，转基因产物表达的下降与对外来人转基因产物(抗人GLA抗体)的体液免疫应答相关(图41)。In plasma, IV administration of AAVhu68.hGLAco (D233C_I359C) resulted in significant levels of transgene product expression (GLA enzyme activity). The average GLA enzyme activity increased approximately 50-fold from day 0 to day 14 after administration (Figure 40). The level of GLA enzyme activity peaked on day 14 and then declined on day 60. At the last time point evaluated (day 60), GLA enzyme activity was approximately 2 times higher than the baseline level observed on day 0. This decrease in transgene product expression after day 14 was expected. Similar to previous NHP studies on AAV delivery of human transgene products, the decrease in transgene product expression was associated with a humoral immune response to foreign human transgene products (anti-human GLA antibodies) (Figure 41).

AAVhu68.hGLAco(D233C_I359C)的静脉内施用也导致治疗后60天心脏、肝脏和肾脏中显著水平的转基因产物表达(GLA酶活性)(图42A)。在心脏和肾脏中观察到GLA酶活性的最大倍数增加(图42B)。在2.5x10¹³GC/kg下，NHP中GLA酶活性的平均增加倍数在心脏、肝脏和肾脏中分别为4.4(437％)、0.5(51％)和3.3(325％)。这些与在2.5x10¹²GC/kg和5.0x10¹²GC/kg的剂量下在KO小鼠中观察到的增加具有相似的量值(图27至图29)，这表明了强大的GL-3清除率(图31A和图31B、图32A、图32B)。图43至图45示出了心脏、肾脏和DRG中的转基因表达(ISH)和转基因产物(IHC)。Intravenous administration of AAVhu68.hGLAco (D233C_I359C) also resulted in significant levels of transgene product expression (GLA enzyme activity) in the heart, liver, and kidney 60 days after treatment ( FIG. 42A ). The greatest fold increases in GLA enzyme activity were observed in the heart and kidney ( FIG. 42B ). At 2.5×10¹³ GC/kg, the mean fold increases in GLA enzyme activity in NHP were 4.4 (437%), 0.5 (51%), and 3.3 (325%) in the heart, liver, and kidney, respectively. These were of similar magnitude to the increases observed in KO mice at doses of 2.5×10¹² GC/kg and 5.0×10¹² GC/kg ( FIGS. 27 to 29 ), indicating robust GL-3 clearance ( FIGS. 31A and 31B , FIG. 32A , FIG. 32B ). Figures 43-45 show transgene expression (ISH) and transgene products (IHC) in heart, kidney, and DRG.

累积地，该研究证实了以2.5x10¹³GC/kg的剂量施用AAVhu68.hGLAco(D233C_I359C)在NHP中是良好耐受的，并且导致用于治疗法布里病(肾脏、心脏、DRG)的靶组织中转基因产物表达(GLA酶活性)的显著增加。Cumulatively, this study demonstrated that administration of AAVhu68.hGLAco(D233C_I359C) at a dose of^2.5x1013 GC/kg was well tolerated in NHPs and resulted in a significant increase in transgene product expression (GLA enzyme activity) in target tissues for the treatment of Fabry disease (kidney, heart, DRG).

实例7：评估食蟹猴静脉内施用AAVhu68.hGLAco(D233C_1359C)的高剂量药理学研究Example 7: High-dose pharmacology study evaluating intravenous administration of AAVhu68.hGLAco(D233C_1359C) in cynomolgus monkeys

成年NHP(N＝3)接受单次IV剂量的AAVhu68.hGLAco(D233C_I359C)，剂量为5.0x10¹³GC/kg。活体评估包括每日进行的临床观察、体重、血液临床病理学(CBC、凝血面板、血清化学[包括心脏生物标志物肌钙蛋白I])和心脏功能评估(ECG和超声心动图)。在载体施用后的第60±3天，对所有动物进行尸检。尸检时，收集组织用于组织病理学检查。收集靶组织用于载体生物分布分析和转基因产物表达定位(人GLA ISH[mRNA]和人GLAIHC[蛋白质])的综合组织学评估。还收集PBMC、脾细胞和肝淋巴细胞以测量对衣壳和转基因产物(IFN-γELIspot)的T细胞应答。Adult NHP (N=3) received a single IV dose of AAVhu68.hGLAco (D233C_I359C) at a dose of 5.0x10¹³ GC/kg. In vivo assessments included daily clinical observations, body weight, blood clinical pathology (CBC, coagulation panel, serum chemistry [including cardiac biomarker troponin I]), and cardiac function assessment (ECG and echocardiogram). All animals were necropsied on day 60±3 after vector administration. At necropsy, tissues were collected for histopathological examination. Target tissues were collected for comprehensive histological assessment of vector biodistribution analysis and transgene product expression localization (human GLA ISH [mRNA] and human GLAIHC [protein]). PBMCs, splenocytes, and liver lymphocytes were also collected to measure T cell responses to capsid and transgene products (IFN-γ ELIspot).

缩写：ADA＝抗药物抗体；BL＝基线；ELISpot＝酶联免疫斑点技术；GLA＝α-半乳糖苷酶A；IFN-γ＝干扰素γ；IHC＝免疫组织化学；IN＝鼻内；ISH＝原位杂交；mRNA＝信使核糖核酸；MS＝质谱；Nab＝中和抗体；PBMC＝外周血单核细胞。Abbreviations: ADA = antidrug antibodies; BL = baseline; ELISpot = enzyme-linked immunospot; GLA = alpha-galactosidase A; IFN-γ = interferon gamma; IHC = immunohistochemistry; IN = intranasal; ISH = in situ hybridization; mRNA = messenger RNA; MS = mass spectrometry; Nab = neutralizing antibody; PBMC = peripheral blood mononuclear cells.

^aBL在给药前长达21天。^a BL up to 21 days prior to dosing.

^b血清化学分析包括肌钙蛋白I(心脏生物标志物)。^b Serum chemistry analysis included troponin I (cardiac biomarker).

^c收集样品并储存以备将来分析。^c Collect samples and store for future analysis.

^d收集组织用于组织病理学、载体生物分布分析和转基因产物表达(人GLA ISH染色[mRNA]和人GLA IHC染色[蛋白质])的组织学评估。收集肝和脾淋巴细胞用于测量对衣壳或转基因产物的T细胞应答(IFN-γELISpot)。^d Tissues were collected for histopathology, vector biodistribution analysis, and histological assessment of transgene product expression (human GLA ISH staining [mRNA] and human GLA IHC staining [protein]). Liver and spleen lymphocytes were collected for measurement of T cell responses to capsid or transgene product (IFN-γ ELISpot).

实例8：在向法布里病小鼠IV施用AAVhu68.hGLAco(D233C_I359C)后的疗效以测定MEDExample 8: Efficacy after IV administration of AAVhu68.hGLAco(D233C_I359C) to Fabry mice to determine the MED

本药理学研究旨在确定AAVhu68.hGLAco(D233C_I359C)IV施用在加重法布里病小鼠模型(Gla KO/TgG3S)中的MED并评估药理学和组织病理学(疗效和安全性)。本研究包括N＝144只动物和两个尸检时间点。评估载体的四个剂量水平。基于小鼠和NHP中的试验疗效数据选择剂量水平。This pharmacology study was designed to determine the MED of AAVhu68.hGLAco(D233C_I359C) IV administered in an exacerbated Fabry disease mouse model (Gla KO/TgG3S) and evaluate pharmacology and histopathology (efficacy and safety). This study included N=144 animals and two necropsy time points. Four dose levels of the vector were evaluated. Dose levels were selected based on experimental efficacy data in mice and NHPs.

如在下表中总结的，以待测定的四个剂量水平之一(5.0x10¹²GC/kg、1.0x10¹³GC/kg、2.5x10¹³GC/kg或5.0x10¹³GC/kg)或媒介物(PBS)向成年(2至3月龄)雄性加重法布里病小鼠(Gla KO/TgG3S)施用AAVhu68.hGLAco(D233C_I359C)。正常雄性WT和WT/TgG3S雄性已被施用媒介物作为对照。雌性加重的Gla KO/TgG3S小鼠也已经接受最高剂量(5.0x10¹³GC/kg)的AAVhu68.hGLAco(D233C_1359C)或媒介物。每组入组16只动物。一半动物(每组8只)将在研究第120天处死，另一半(每组8只)将在至少80％的媒介物处理的Gla KO/TgG3S小鼠已经达到人道终点(定义为引起行走障碍和/或体重下降≥20％的峰值体重的严重震颤和共济失调)时进行尸检。As summarized in the table below, adult (2 to 3 months old) male aggravated Fabry mice (Gla KO/TgG3S) were administered AAVhu68.hGLAco (D233C_I359C) at one of the four dose levels to be tested (5.0x10¹² GC/kg, 1.0x10¹³ GC/kg, 2.5x10¹³ GC/kg or 5.0x10¹³ GC/kg) or vehicle (PBS). Normal male WT and WT/TgG3S males have been administered vehicle as controls. Female aggravated Gla KO/TgG3S mice have also received the highest dose (5.0x10¹³ GC/kg) of AAVhu68.hGLAco (D233C_1359C) or vehicle. 16 animals were enrolled in each group. Half of the animals (8 per group) will be sacrificed on study day 120, and the other half (8 per group) will be necropsied when at least 80% of vehicle-treated Gla KO/TgG3S mice have reached humane endpoints (defined as severe tremor and ataxia causing walking impairment and/or weight loss ≥20% of peak body weight).

缩与：F＝雌性；Gla＝α-半乳糖苷酶A(基因)；IV＝静脉内；KO＝敲除；M＝雄性；N/A＝不适用；ROA＝施用途径；TBD＝待确定；WT＝野生型。Abbreviations: F = female; Gla = α-galactosidase A (gene); IV = intravenous; KO = knockout; M = male; N/A = not applicable; ROA = route of administration; TBD = to be determined; WT = wild type.

活体评估包括每天进行的活力检查以监测存活率、体重测量、临床观察、热感觉功能评估(热板潜伏期)、血清尿素氮(BUN)水平、尿渗透压、尿量和血清转基因表达(GLA酶活性)的评估。在人道终点和较早的时间点(第120天)进行尸检。尸检时，收集一份完整的组织清单用于组织病理学评估。收集另外的组织(DRG、心脏、肾脏)以评估GL-3储存(GL-3IHC)。还收集靶组织用于转基因表达测定(GLA酶活性)和通过LC-MS/MS定量GL-3(大脑、心脏、肾脏、肝脏、大肠)。收集血液用于CBC/分类和血清临床化学分析，包括BUN水平。还收集血浆以评估转基因产物表达(GLA酶活性)和lyso-Gb₃储存。In vivo assessment includes daily vitality check to monitor survival rate, body weight measurement, clinical observation, thermal sensory function assessment (hot plate latency), serum urea nitrogen (BUN) level, urine osmotic pressure, urine volume and serum transgenic expression (GLA enzyme activity) assessment. Autopsy is performed at humane endpoint and earlier time point (120th day). During autopsy, a complete list of tissues is collected for histopathological assessment. Other tissues (DRG, heart, kidney) are collected to assess GL-3 storage (GL-3IHC). Target tissues are also collected for transgenic expression determination (GLA enzyme activity) and quantitative GL-3 (brain, heart, kidney, liver, large intestine) by LC-MS/MS. Blood is collected for CBC/classification and serum clinical chemistry analysis, including BUN level. Plasma is also collected to assess transgenic product expression (GLA enzyme activity) and lyso-Gb₃ storage.

进行活体体重评估和热板试验的人员对每只小鼠的处理条件和基因型不知情。Personnel performing the in vivo body weight assessments and hot plate assays were blinded to the treatment conditions and genotype of each mouse.

基于存活益处、体重、热感觉功能(使用热板测定法评估)、肾功能(通过BUN水平、尿量和尿渗透压评估)、靶组织中GL-3溶酶体贮积的校正和疾病相关靶器官中的转基因产物表达(GAL活性水平)的分析来确定MED。The MED was determined based on analysis of survival benefit, body weight, thermal sensory function (assessed using a hot plate assay), renal function (assessed by BUN levels, urine volume, and urine osmolality), correction of GL-3 lysosomal storage in target tissues, and transgene product expression (GAL activity levels) in disease-relevant target organs.

实例9：AAVhu68.hGLAco(D233C_I359C)对成年非人灵长类动物静脉内施用的毒理学研究Example 9: Toxicology study of intravenous administration of AAVhu68.hGLAco(D233C_I359C) to adult non-human primates

进行一项为期180天的GLP合规毒理学研究以评估AAVhu68.hGLAco(D233C_I359C)在低剂量(1.0x10¹³GC/kg)、中剂量(2.5x10¹³GC/kg)或高剂量(5.0x10¹³GC/kg)下单次IV施用于食蟹猴NHP后的安全性、耐受性、转基因产物表达、生物分布和排泄情况。另外的NHP被施用媒介物(PBS)作为对照。A 180-day GLP-compliant toxicology study was conducted to evaluate the safety, tolerability, transgene product expression, biodistribution, and excretion of AAVhu68.hGLAco(D233C_I359C) at low (1.0x10¹³ GC/kg), medium (2.5x10¹³ GC/kg), or high (5.0x10¹³ GC/kg) doses after a single IV administration to cynomolgus monkey NHPs. Additional NHPs were administered vehicle (PBS) as a control.

选择NHP(食蟹猴)用于计划的毒理学研究。选择该模型是因为我们对AAV载体在NHP中的应用有丰富的经验，并且NHP的毒理学和免疫应答接近地代表了人的毒理学和免疫应答。选择成年NHP(2至8岁)代表计划临床试验的患者群体。研究中将包括雄性和雌性。NHP (crab-eating monkey) was selected for the planned toxicology study. This model was selected because we have extensive experience with the use of AAV vectors in NHP, and the toxicology and immune responses of NHP closely represent those of humans. Adult NHP (2 to 8 years old) were selected to represent the patient population for the planned clinical trial. Both males and females will be included in the study.

选择IV途径是因为全身施用在疾病相关靶组织(DRG、肾脏和心脏)和非疾病相关(肝脏)靶组织中提供最佳转导和转基因产物表达。The IV route was chosen because systemic administration provides optimal transduction and transgene product expression in both disease-relevant target tissues (DRG, kidney, and heart) and non-disease-relevant (liver) target tissues.

表——NHP毒理学研究组Table - NHP Toxicology Research Group

在整个研究过程中，以频繁的时间间隔对生命体征、体重、血液临床病理学(CBC与差值、临床化学、凝血面板)和CSF(细胞学和化学)进行笼侧临床观察和评估。监测全血细胞计数(CBC)、肝脏参数和补体活化，因为急性肝毒性、血小板减少症和补体活化是全身性AAV施用后的已知毒性。Cage-side clinical observations and assessments of vital signs, body weight, blood clinical pathology (CBC with differential, clinical chemistry, coagulation panel), and CSF (cytology and chemistry) were performed at frequent intervals throughout the study. Complete blood count (CBC), liver parameters, and complement activation were monitored, as acute hepatotoxicity, thrombocytopenia, and complement activation are known toxicities following systemic AAV administration.

肌钙蛋白I测试作为临床病理学小组的一部分，与基线和治疗后每30天进行的超声心动图评估一起包括在内，以监测心脏毒性的迹象，因为AAVhu68在IV施用后表现出对心脏组织的高度嗜性。Troponin I testing was included as part of the clinical pathology panel along with echocardiographic assessments performed at baseline and every 30 days after treatment to monitor for signs of cardiotoxicity, as AAVhu68 exhibits a high tropism for cardiac tissue following IV administration.

在基线、第14天、第28天和此后每30天进行神经病学检查。在基线、第28天、第60天和第180天进行双侧正中神经的感觉神经传导研究(NCS)以监测DRG感觉神经元变性的迹象。基于NHP中感觉神经元变性的已知动力学选择这些时间点，这些动力学在载体施用后14至21天出现，并且到第30天在正中神经NCS上可检测。对于神经病学检查，评估分为五个部分，评估精神状态、姿势和步态、本体感受、颅神经和脊柱反射。每次评估的测试都按相同的顺序进行。神经病学检查评估员对处理组无正式设盲；然而，评估员在评估时通常不知道处理组。在适用的情况下，对每个评估类别给出数值评分并记录(止常：1；异常：2；下降：3；增加：4；无：5；N/A：不适用)。对于感觉NCS，使用Nicolet系统(Natus神经学公司)和分析软件测量感觉神经动作电位振幅和传导速度。进行NCS分析的评估员对处理组正式设盲。Neurological examinations were performed at baseline, day 14, day 28, and every 30 days thereafter. Sensory nerve conduction studies (NCS) of bilateral median nerves were performed at baseline, day 28, day 60, and day 180 to monitor for signs of DRG sensory neuron degeneration. These time points were selected based on the known kinetics of sensory neuron degeneration in NHPs, which occur 14 to 21 days after vehicle administration and are detectable on median nerve NCS by day 30. For neurological examinations, the assessment was divided into five parts, assessing mental state, posture and gait, proprioception, cranial nerves, and spinal reflexes. Tests were performed in the same order for each assessment. Neurological examination assessors were not formally blinded to the treatment group; however, assessors were generally unaware of the treatment group at the time of the assessment. Where applicable, a numerical score was given and recorded for each assessment category (normal: 1; abnormal: 2; decreased: 3; increased: 4; none: 5; N/A: not applicable). For sensory NCS, a Nicolet Systems (Natus Neurological Corporation) and The analysis software measured sensory nerve action potential amplitude and conduction velocity. The assessors performing the NCS analysis were formally blinded to the treatment groups.

在血清中测量转基因产物的表达(GLA酶活性)。在转基因表达的预期开始、峰值和平台期间以频繁的间隔收集样品。同样在相应时间点使用酶联免疫吸附测定(ELISA)在血清中评估抗转基因产物抗体(即，抗药物抗体[ADA])，以评估对可能全身性发生的外来人类转基因产物的潜在抗体应答。The expression of the transgenic product (GLA enzyme activity) is measured in serum. Samples are collected at frequent intervals during the expected onset, peak, and platform of transgenic expression. Anti-transgenic product antibodies (i.e., anti-drug antibodies [ADA]) are also assessed in serum at the corresponding time points using enzyme-linked immunosorbent assay (ELISA) to assess potential antibody responses to foreign human transgenic products that may occur systemically.

在基线测量针对AAVhu68衣壳的中和抗体应答以评估对载体转导(生物分布)的影响，然后每月评估NAb应答的动力学。收集外周血单核细胞以使用IFN-γELISpot测定评估对衣壳和/或转基因产物的T细胞应答。选择PBMC收集的时间点，因为T细胞和B细胞免疫应答通常在30天内发生在NHP中。在尸检时，还收集来自脾和肝的组织驻留淋巴细胞，用于评估对衣壳和/或转基因产物的T细胞应答。Neutralizing antibody responses to AAVhu68 capsid were measured at baseline to assess the effect on vector transduction (biodistribution), and then monthly to assess the kinetics of NAb responses. Peripheral blood mononuclear cells were collected to assess T cell responses to capsid and/or transgene product using IFN-γ ELISpot assay. The time point for PBMC collection was chosen because T cell and B cell immune responses typically occur in NHPs within 30 days. At necropsy, tissue-resident lymphocytes from the spleen and liver were also collected for assessment of T cell responses to capsid and/or transgene product.

收集血清和CSF以评估载体分布，收集尿液和粪便以评估载体排泄(脱落)。在频繁的时间点收集这些样品并通过定量聚合酶链式反应(qPCR)进行定量以能够评估处理后载体分布和排泄的动力学。还收集CSF和血清样品并存档，以备将来分析，以防任何发现需要分析。Serum and CSF were collected to assess vector distribution, and urine and feces were collected to assess vector excretion (shedding). These samples were collected at frequent time points and quantified by quantitative polymerase chain reaction (qPCR) to be able to assess the kinetics of vector distribution and excretion after treatment. CSF and serum samples were also collected and archived for future analysis in case any findings require analysis.

在第180天尸检时，收集一份完整的组织清单用于组织病理学和载体生物分布分析。还收集组织以评估转基因产物表达。选择所有组织以包括法布里病的可能靶组织(肾脏、心脏、DRG、肠)和/或高度灌注的外周器官(如肝脏和肾脏)。此外，从肝脏、脾脏和骨髓中采集淋巴细胞，以评估尸检时这些器官中对衣壳和/或转基因产物有反应性的T细胞的存在。At the 180th day autopsy, a complete list of tissues was collected for histopathology and vector biodistribution analysis. Tissues were also collected to assess transgenic product expression. All tissues were selected to include possible target tissues for Fabry disease (kidney, heart, DRG, intestine) and/or highly perfused peripheral organs (such as liver and kidney). In addition, lymphocytes were collected from the liver, spleen, and bone marrow to assess the presence of T cells reactive to capsid and/or transgenic product in these organs at autopsy.

(独立文本的序列表)(Sequence listing for stand-alone text)

以下信息是为数字标识符<223>下含独立文本的序列提供的。The following information is provided for sequences containing stand-alone text under the numeric identifier <223>.

本说明书中引用的所有专利和非专利出版物通过引用整体并入本文。国际专利申请号PCT/US2019/05567，申请日为2019年10月10日，美国临时专利申请第63/089,850号，申请日为2020年10月9日，美国临时专利申请第63/146,286号，申请日为2021年2月5日，美国临时专利申请第63/186,092号，申请日为2021年5月8日，其全部内容通过引用并入本文。本文提交的名为“19-8855PCT_ST25.txt”的序列表和其中的序列和文本通过引用并入本文。尽管已经参照具体实施例描述了本发明，但是应当理解，在不脱离本发明的精神的情况下可以进行修改。这些修改旨在落入所附权利要求的范围内。All patents and non-patent publications cited in this specification are incorporated herein by reference in their entirety. International Patent Application No. PCT/US2019/05567, filed on October 10, 2019, U.S. Provisional Patent Application No. 63/089,850, filed on October 9, 2020, U.S. Provisional Patent Application No. 63/146,286, filed on February 5, 2021, and U.S. Provisional Patent Application No. 63/186,092, filed on May 8, 2021, are incorporated herein by reference in their entirety. The sequence table entitled "19-8855PCT_ST25.txt" submitted herein and the sequences and texts therein are incorporated herein by reference. Although the present invention has been described with reference to specific embodiments, it should be understood that modifications may be made without departing from the spirit of the present invention. These modifications are intended to fall within the scope of the appended claims.

序列表Sequence Listing

<110> 宾夕法尼亚大学董事会（The Trustees of the University ofPennsylvania）<110> The Trustees of the University of Pennsylvania

<120> 用于治疗法布里病的组合物和方法<120> Compositions and methods for treating Fabry disease

<130> UPN-19-8855.PCT<130> UPN-19-8855.PCT

<150> US 63/089,850<150> US 63/089,850

<151> 2020-10-09<151> 2020-10-09

<150> US 63/146,286<150> US 63/146,286

<151> 2021-02-05<151> 2021-02-05

<150> US 63/186,092<150> US 63/186,092

<151> 2021-05-08<151> 2021-05-08

<160> 32<160> 32

<170> PatentIn版本3.5<170> PatentIn version 3.5

<210> 1<210> 1

<211> 1287<211> 1287

<212> DNA<212> DNA

<213> 智人<213> Homo sapiens

<220><220>

<221> CDS<221> CDS

<222> (1)..(1287)<222> (1)..(1287)

<400> 1<400> 1

atg cag ctg agg aac cca gaa cta cat ctg ggc tgc gcg ctt gcg ctt 48atg cag ctg agg aac cca gaa cta cat ctg ggc tgc gcg ctt gcg ctt 48

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 151 5 10 15

cgc ttc ctg gcc ctc gtt tcc tgg gac atc cct ggg gct aga gca ctg 96cgc ttc ctg gcc ctc gtt tcc tgg gac atc cct ggg gct aga gca ctg 96

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 3020 25 30

gac aat gga ttg gca agg acg cct acc atg ggc tgg ctg cac tgg gag 144gac aat gga ttg gca agg acg cct acc atg ggc tgg ctg cac tgg gag 144

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 4535 40 45

cgc ttc atg tgc aac ctt gac tgc cag gaa gag cca gat tcc tgc atc 192cgc ttc atg tgc aac ctt gac tgc cag gaa gag cca gat tcc tgc atc 192

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleArg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 6050 55 60

agt gag aag ctc ttc atg gag atg gca gag ctc atg gtc tca gaa ggc 240agt gag aag ctc ttc atg gag atg gca gag ctc atg gtc tca gaa ggc 240

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlySer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 8065 70 75 80

tgg aag gat gca ggt tat gag tac ctc tgc att gat gac tgt tgg atg 288tgg aag gat gca ggt tat gag tac ctc tgc att gat gac tgt tgg atg 288

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 9585 90 95

gct ccc caa aga gat tca gaa ggc aga ctt cag gca gac cct cag cgc 336gct ccc caa aga gat tca gaa ggc aga ctt cag gca gac cct cag cgc 336

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110100 105 110

ttt cct cat ggg att cgc cag cta gct aat tat gtt cac agc aaa gga 384ttt cct cat ggg att cgc cag cta gct aat tat gtt cac agc aaa gga 384

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125115 120 125

ctg aag cta ggg att tat gca gat gtt gga aat aaa acc tgc gca ggc 432ctg aag cta ggg att tat gca gat gtt gga aat aaa acc tgc gca ggc 432

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140130 135 140

ttc cct ggg agt ttt gga tac tac gac att gat gcc cag acc ttt gct 480ttc cct ggg agt ttt gga tac tac gac att gat gcc cag acc ttt gct 480

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160145 150 155 160

gac tgg gga gta gat ctg cta aaa ttt gat ggt tgt tac tgt gac agt 528gac tgg gga gta gat ctg cta aaa ttt gat ggt tgt tac tgt gac agt 528

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175165 170 175

ttg gaa aat ttg gca gat ggt tat aag cac atg tcc ttg gcc ctg aat 576ttg gaa aat ttg gca gat ggt tat aag cac atg tcc ttg gcc ctg aat 576

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190180 185 190

agg act ggc aga agc att gtg tac tcc tgt gag tgg cct ctt tat atg 624agg act ggc aga agc att gtg tac tcc tgt gag tgg cct ctt tat atg 624

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205195 200 205

tgg ccc ttt caa aag ccc aat tat aca gaa atc cga cag tac tgc aat 672tgg ccc ttt caa aag ccc aat tat aca gaa atc cga cag tac tgc aat 672

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220210 215 220

cac tgg cga aat ttt gct gac att gat gat tcc tgg aaa agt ata aag 720cac tgg cga aat ttt gct gac att gat gat tcc tgg aaa agt ata aag 720

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile LysHis Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240225 230 235 240

agt atc ttg gac tgg aca tct ttt aac cag gag aga att gtt gat gtt 768agt atc ttg gac tgg aca tct ttt aac cag gag aga att gtt gat gtt 768

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255245 250 255

gct gga cca ggg ggt tgg aat gac cca gat atg tta gtg att ggc aac 816gct gga cca ggg ggt tgg aat gac cca gat atg tta gtg att ggc aac 816

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270260 265 270

ttt ggc ctc agc tgg aat cag caa gta act cag atg gcc ctc tgg gct 864ttt ggc ctc agc tgg aat cag caa gta act cag atg gcc ctc tgg gct 864

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285275 280 285

atc atg gct gct cct tta ttc atg tct aat gac ctc cga cac atc agc 912atc atg gct gct cct tta ttc atg tct aat gac ctc cga cac atc agc 912

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300290 295 300

cct caa gcc aaa gct ctc ctt cag gat aag gac gta att gcc atc aat 960cct caa gcc aaa gct ctc ctt cag gat aag gac gta att gcc atc aat 960

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320305 310 315 320

cag gac ccc ttg ggc aag caa ggg tac cag ctt aga cag gga gac aac 1008cag gac ccc ttg ggc aag caa ggg tac cag ctt aga cag gga gac aac 1008

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335325 330 335

ttt gaa gtg tgg gaa cga cct ctc tca ggc tta gcc tgg gct gta gct 1056ttt gaa gtg tgg gaa cga cct ctc tca ggc tta gcc tgg gct gta gct 1056

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350340 345 350

atg ata aac cgg cag gag att ggt gga cct cgc tct tat acc atc gca 1104atg ata aac cgg cag gag att ggt gga cct cgc tct tat acc atc gca 1104

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile AlaMet Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365355 360 365

gtt gct tcc ctg ggt aaa gga gtg gcc tgt aat cct gcc tgc ttc atc 1152gtt gct tcc ctg ggt aaa gga gtg gcc tgt aat cct gcc tgc ttc atc 1152

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380370 375 380

aca cag ctc ctc cct gtg aaa agg aag cta ggg ttc tat gaa tgg act 1200aca cag ctc ctc cct gtg aaa agg aag cta ggg ttc tat gaa tgg act 1200

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400385 390 395 400

tca agg tta aga agt cac ata aat ccc aca ggc act gtt ttg ctt cag 1248tca agg tta aga agt cac ata aat ccc aca ggc act gtt ttg ctt cag 1248

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415405 410 415

cta gaa aat aca atg cag atg tca tta aaa gac tta ctt 1287cta gaa aat aca atg cag atg tca tta aaa gac tta ctt 1287

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu LeuLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425420 425

<210> 2<210> 2

<211> 429<211> 429

<212> PRT<212> PRT

<213> 智人<213> Homo sapiens

<400> 2<400> 2

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 151 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 3020 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 4535 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleArg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 6050 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlySer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 8065 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 9585 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile LysHis Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile AlaMet Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu LeuLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425420 425

<210> 3<210> 3

<211> 1287<211> 1287

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 3<400> 3

atgcagctga gaaatcccga gctgcacctg ggctgtgccc tggctctgag atttctggcc 60atgcagctga gaaatcccga gctgcacctg ggctgtgccc tggctctgag atttctggcc 60

ctggtgtctt gggacatccc tggcgctaga gccctggata acggcctggc cagaacacct 120ctggtgtctt gggacatccc tggcgctaga gccctggata acggcctggc cagaacacct 120

acaatgggct ggctgcactg ggagagattc atgtgcaacc tggactgcca agaggaaccc 180acaatgggct ggctgcactg ggagagattc atgtgcaacc tggactgcca agaggaaccc 180

gacagctgca tcagcgagaa gctgttcatg gaaatggccg agctgatggt gtccgaaggc 240gacagctgca tcagcgagaa gctgttcatg gaaatggccg agctgatggt gtccgaaggc 240

tggaaggacg ccggctacga gtacctgtgc atcgacgact gttggatggc ccctcagaga 300tggaaggacg ccggctacga gtacctgtgc atcgacgact gttggatggc ccctcagaga 300

gactctgagg gcagactgca ggccgatcct cagagatttc cccacggcat tagacagctg 360gactctgagg gcagactgca ggccgatcct cagagatttc cccacggcat tagacagctg 360

gccaactacg tgcacagcaa gggcctgaag ctgggcatct acgccgacgt gggcaacaag 420gccaactacg tgcacagcaa gggcctgaag ctgggcatct acgccgacgt gggcaacaag 420

acctgtgccg gctttcctgg cagcttcggc tactacgata tcgacgccca gaccttcgcc 480acctgtgccg gctttcctgg cagcttcggc tactacgata tcgacgccca gaccttcgcc 480

gattggggag tcgatctgct gaagttcgac ggctgctact gcgacagcct ggaaaatctg 540gattggggag tcgatctgct gaagttcgac ggctgctact gcgacagcct ggaaaatctg 540

gccgacggct acaagcacat gtcactggcc ctgaatcgga ccggccgcag catcgtgtac 600gccgacggct acaagcacat gtcactggcc ctgaatcgga ccggccgcag catcgtgtac 600

tcttgcgagt ggcccctgta tatgtggccc ttccagaagc ctaactacac cgagatcaga 660tcttgcgagt ggcccctgta tatgtggccc ttccagaagc ctaactacac cgagatcaga 660

cagtactgca accactggcg gaacttcgcc gacatcgacg atagctggaa gtccatcaag 720cagtactgca accactggcg gaacttcgcc gacatcgacg atagctggaa gtccatcaag 720

agcatcctgg actggaccag cttcaatcaa gagcggatcg tggacgtggc aggacctggc 780agcatcctgg actggaccag cttcaatcaa gagcggatcg tggacgtggc aggacctggc 780

ggatggaacg atcctgacat gctggtcatc ggcaacttcg gcctgagctg gaaccagcaa 840ggatggaacg atcctgacat gctggtcatc ggcaacttcg gcctgagctg gaaccagcaa 840

gtgacccaga tggccctgtg ggccattatg gccgctcctc tgttcatgag caacgacctg 900gtgacccaga tggccctgtg ggccattatg gccgctcctc tgttcatgag caacgacctg 900

agacacatca gccctcaggc caaggctctg ctgcaggaca aggatgtgat cgctatcaac 960agacacatca gccctcaggc caaggctctg ctgcaggaca aggatgtgat cgctatcaac 960

caggatcctc tgggcaagca gggctaccag ctgagacagg gcgacaattt cgaagtgtgg 1020caggatcctc tgggcaagca gggctaccag ctgagacagg gcgacaattt cgaagtgtgg 1020

gaaagacccc tgagcggact ggcttgggcc gtcgccatga tcaacagaca agagatcggc 1080gaaagacccc tgagcggact ggcttgggcc gtcgccatga tcaacagaca agagatcggc 1080

ggaccccggt cctacacaat tgccgtggct tctctcggca aaggcgtggc ctgtaatccc 1140ggaccccggt cctacacaat tgccgtggct tctctcggca aaggcgtggc ctgtaatccc 1140

gcctgcttta tcacacagct gctgcccgtg aagagaaagc tgggctttta cgagtggacc 1200gcctgcttta tcacacagct gctgcccgtg aagagaaagc tgggctttta cgagtggacc 1200

agcagactgc ggagccacat caatcctacc ggcacagtgc tgctgcagct ggaaaacaca 1260agcagactgc ggagccacat caatcctacc ggcacagtgc tgctgcagct ggaaaacaca 1260

atgcagatga gcctgaagga cctgctg 1287atgcagatga gcctgaagga cctgctg 1287

<210> 4<210> 4

<211> 1287<211> 1287

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 4<400> 4

atgcaactga gaaatcctga actgcacctg ggctgcgccc tggctctgag atttctggct 60atgcaactga gaaatcctga actgcacctg ggctgcgccc tggctctgag atttctggct 60

ctggtgtcct gggacatccc tggcgctaga gccctggata acggcctggc cagaacacct 120ctggtgtcct gggacatccc tggcgctaga gccctggata acggcctggc cagaacacct 120

acaatgggct ggctgcactg ggagagattc atgtgcaacc tggactgcca agaggaaccc 180acaatgggct ggctgcactg ggagagattc atgtgcaacc tggactgcca agaggaaccc 180

gacagctgca tcagcgagaa gctgttcatg gaaatggccg agctgatggt gtccgaaggc 240gacagctgca tcagcgagaa gctgttcatg gaaatggccg agctgatggt gtccgaaggc 240

tggaaggacg ccggctacga gtacctgtgc atcgacgact gttggatggc ccctcagaga 300tggaaggacg ccggctacga gtacctgtgc atcgacgact gttggatggc ccctcagaga 300

gactctgagg gcagactgca ggccgatcct cagagatttc cccacggcat tagacagctg 360gactctgagg gcagactgca ggccgatcct cagagatttc cccacggcat tagacagctg 360

gccaactacg tgcacagcaa gggcctgaag ctgggcatct acgccgacgt gggcaacaag 420gccaactacg tgcacagcaa gggcctgaag ctgggcatct acgccgacgt gggcaacaag 420

acctgtgccg gctttcctgg cagcttcggc tactacgata tcgacgccca gaccttcgcc 480acctgtgccg gctttcctgg cagcttcggc tactacgata tcgacgccca gaccttcgcc 480

gattggggag tcgatctgct gaagttcgac ggctgctact gcgacagcct ggaaaatctg 540gattggggag tcgatctgct gaagttcgac ggctgctact gcgacagcct ggaaaatctg 540

gccgacggct acaagcacat gtctctggcc ctgaatcgga ccggcagatc catcgtgtac 600gccgacggct acaagcacat gtctctggcc ctgaatcgga ccggcagatc catcgtgtac 600

agctgcgagt ggcccctgta catgtggccc ttccagaagc ctaactacac cgagatcaga 660agctgcgagt ggcccctgta catgtggccc ttccagaagc ctaactacac cgagatcaga 660

cagtactgca accactggcg gaacttcgcc gacatctgcg atagctggaa gtccatcaag 720cagtactgca accactggcg gaacttcgcc gacatctgcg atagctggaa gtccatcaag 720

agcatcctgg actggaccag cttcaatcaa gagcggatcg tggacgtggc aggacctggc 780agcatcctgg actggaccag cttcaatcaa gagcggatcg tggacgtggc aggacctggc 780

ggatggaacg atcctgacat gctggtcatc ggcaacttcg gcctgagctg gaaccagcaa 840ggatggaacg atcctgacat gctggtcatc ggcaacttcg gcctgagctg gaaccagcaa 840

gtgacccaga tggccctgtg ggccattatg gccgctcctc tgttcatgag caacgacctg 900gtgacccaga tggccctgtg ggccattatg gccgctcctc tgttcatgag caacgacctg 900

agacacatca gccctcaggc caaggctctg ctgcaggaca aggatgtgat cgctatcaac 960agacacatca gccctcaggc caaggctctg ctgcaggaca aggatgtgat cgctatcaac 960

caggatcctc tgggcaagca gggctaccag ctgagacagg gcgacaattt cgaagtgtgg 1020caggatcctc tgggcaagca gggctaccag ctgagacagg gcgacaattt cgaagtgtgg 1020

gaaagacccc tgagcggact ggcttgggcc gtcgccatga tcaaccggca agagtgcggc 1080gaaagacccc tgagcggact ggcttgggcc gtcgccatga tcaaccggca agagtgcggc 1080

ggccccagat cctacacaat cgccgtggcc agtctcggca aaggcgtggc atgtaatccc 1140ggccccagat cctacacaat cgccgtggcc agtctcggca aaggcgtggc atgtaatccc 1140

gcctgcttca tcacacagct gctgcccgtg aagagaaagc tgggctttta cgagtggacc 1200gcctgcttca tcacacagct gctgcccgtg aagagaaagc tgggctttta cgagtggacc 1200

agcagactgc ggagccacat caatcctacc ggcacagtgc tgctgcagct ggaaaacacc 1260agcagactgc ggagccacat caatcctacc ggcacagtgc tgctgcagct ggaaaacacc 1260

atgcagatga gcctgaagga cctgctg 1287atgcagatga gcctgaagga cctgctg 1287

<210> 5<210> 5

<211> 1287<211> 1287

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 5<400> 5

atgcaactga gaaatcctga actgcacctg ggctgcgccc tggctctgag atttctggct 60atgcaactga gaaatcctga actgcacctg ggctgcgccc tggctctgag atttctggct 60

ctggtgtcct gggacatccc tggcgctaga gccctggata acggcctggc cagaacacct 120ctggtgtcct gggacatccc tggcgctaga gccctggata acggcctggc cagaacacct 120

acaatgggct ggctgcactg ggagagattc tgctgcaacc tggactgcca agaggaaccc 180acaatgggct ggctgcactg ggagagattc tgctgcaacc tggactgcca agaggaaccc 180

gacagctgca tcagcgagaa gctgttcatg gaaatggccg agctgatggt gtccgaaggc 240gacagctgca tcagcgagaa gctgttcatg gaaatggccg agctgatggt gtccgaaggc 240

tggaaggacg ccggctacga gtacctgtgc atcgacgact gttggatggc ccctcagaga 300tggaaggacg ccggctacga gtacctgtgc atcgacgact gttggatggc ccctcagaga 300

gactctgagg gcagactgca ggccgatcct cagagatttc cccacggcat tagacagctg 360gactctgagg gcagactgca ggccgatcct cagagatttc cccacggcat tagacagctg 360

gccaactacg tgcacagcaa gggcctgaag ctgggcatct acgccgacgt gggcaacaag 420gccaactacg tgcacagcaa gggcctgaag ctgggcatct acgccgacgt gggcaacaag 420

acctgtgccg gctttcctgg cagcttcggc tactacgata tcgacgccca gaccttcgcc 480acctgtgccg gctttcctgg cagcttcggc tactacgata tcgacgccca gaccttcgcc 480

gattggggag tcgatctgct gaagttcgac ggctgctact gcgacagcct ggaaaatctg 540gattggggag tcgatctgct gaagttcgac ggctgctact gcgacagcct ggaaaatctg 540

gccgacggct acaagcacat gtctctggcc ctgaatcgga ccggcagatc catcgtgtac 600gccgacggct acaagcacat gtctctggcc ctgaatcgga ccggcagatc catcgtgtac 600

agctgcgagt ggcccctgta catgtggccc ttccagaagc ctaactacac cgagatcaga 660agctgcgagt ggcccctgta catgtggccc ttccagaagc ctaactacac cgagatcaga 660

cagtactgca accactggcg gaacttcgcc gacatcgacg atagctggaa gtccatcaag 720cagtactgca accactggcg gaacttcgcc gacatcgacg atagctggaa gtccatcaag 720

agcatcctgg actggaccag cttcaatcaa gagcggatcg tggacgtggc aggacctggc 780agcatcctgg actggaccag cttcaatcaa gagcggatcg tggacgtggc aggacctggc 780

ggatggaacg atcctgacat gctggtcatc ggcaacttcg gcctgagctg gaaccagcaa 840ggatggaacg atcctgacat gctggtcatc ggcaacttcg gcctgagctg gaaccagcaa 840

gtgacccaga tggccctgtg ggccattatg gccgctcctc tgttcatgag caacgacctg 900gtgacccaga tggccctgtg ggccattatg gccgctcctc tgttcatgag caacgacctg 900

agacacatca gccctcaggc caaggctctg ctgcaggaca aggatgtgat cgctatcaac 960agacacatca gccctcaggc caaggctctg ctgcaggaca aggatgtgat cgctatcaac 960

caggatcctc tgggcaagca gggctaccag ctgagacagg gcgacaattt cgaagtgtgg 1020caggatcctc tgggcaagca gggctaccag ctgagacagg gcgacaattt cgaagtgtgg 1020

gaaagacccc tgagcggact ggcttgggcc gtcgccatga tcaaccggca agagatttgc 1080gaaagacccc tgagcggact ggcttgggcc gtcgccatga tcaaccggca agagatttgc 1080

ggccccagat cctacacaat cgccgtggcc agtctcggca aaggcgtggc atgtaatccc 1140ggccccagat cctacacaat cgccgtggcc agtctcggca aaggcgtggc atgtaatccc 1140

gcctgcttca tcacacagct gctgcccgtg aagagaaagc tgggctttta cgagtggacc 1200gcctgcttca tcacacagct gctgcccgtg aagagaaagc tgggctttta cgagtggacc 1200

agcagactgc ggagccacat caatcctacc ggcacagtgc tgctgcagct ggaaaacacc 1260agcagactgc ggagccacat caatcctacc ggcacagtgc tgctgcagct ggaaaacacc 1260

atgcagatga gcctgaagga cctgctg 1287atgcagatga gcctgaagga cctgctg 1287

<210> 6<210> 6

<211> 4297<211> 4297

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> CB7.CI.hGLAco(D233C/I359C).WPRE.rBG<223> CB7.CI.hGLAco(D233C/I359C).WPRE.rBG

<220><220>

<221> repeat_region<221> repeat_region

<222> (1)..(130)<222> (1)..(130)

<223> 5' ITR<223> 5' ITR

<220><220>

<221> misc_feature<221> misc_feature

<222> (198)..(579)<222> (198)..(579)

<223> CMV立即早期增强子<223> CMV immediate early enhancer

<220><220>

<221> promoter<221> promoter

<222> (582)..(863)<222> (582)..(863)

<223> CB启动子<223> CB promoter

<220><220>

<221> Intron<221> Intron

<222> (956)..(1928)<222> (956)..(1928)

<223> 鸡β-肌动蛋白内含子<223> Chicken β-actin intron

<220><220>

<221> CDS<221> CDS

<222> (1948)..(3240)<222> (1948)..(3240)

<223> hGLAco.D233C.I359C<223> hGLAco.D233C.I359C

<220><220>

<221> misc_feature<221> misc_feature

<222> (2353)..(2372)<222> (2353)..(2372)

<223> P18<223> P18

<220><220>

<221> misc_feature<221> misc_feature

<222> (2644)..(2646)<222> (2644)..(2646)

<223> D233C<223> D233C

<220><220>

<221> misc_feature<221> misc_feature

<222> (3022)..(3024)<222> (3022)..(3024)

<223> I359C<223> I359C

<220><220>

<221> misc_feature<221> misc_feature

<222> (3253)..(3841)<222> (3253)..(3841)

<223> WPRE<223> WPRE

<220><220>

<221> misc_feature<221> misc_feature

<222> (3609)..(3628)<222> (3609)..(3628)

<223> P19<223> P19

<220><220>

<221> polyA_signal<221> polyA_signal

<222> (3953)..(4079)<222> (3953)..(4079)

<223> 兔珠蛋白poly A<223> Rabbit globin poly A

<220><220>

<221> repeat_region<221> repeat_region

<222> (4168)..(4297)<222> (4168)..(4297)

<223> 3' ITR<223> 3' ITR

<400> 6<400> 6

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960

cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440

ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1800tctccagcct cggggctgtc cgcgggggga cggctgcctt cggggggggac ggggcagggc 1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920

tggcaaagaa ttcgcggccg cgccacc atg caa ctg aga aat cct gaa ctg cac 1974tggcaaagaa ttcgcggccg cgccacc atg caa ctg aga aat cct gaa ctg cac 1974

Met Gln Leu Arg Asn Pro Glu Leu HisMet Gln Leu Arg Asn Pro Glu Leu His

1 51 5

ctg ggc tgc gcc ctg gct ctg aga ttt ctg gct ctg gtg tcc tgg gac 2022ctg ggc tgc gcc ctg gct ctg aga ttt ctg gct ctg gtg tcc tgg gac 2022

Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp AspLeu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp

10 15 20 2510 15 20 25

atc cct ggc gct aga gcc ctg gat aac ggc ctg gcc aga aca cct aca 2070atc cct ggc gct aga gcc ctg gat aac ggc ctg gcc aga aca cct aca 2070

Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro ThrIle Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr

30 35 4030 35 40

atg ggc tgg ctg cac tgg gag aga ttc atg tgc aac ctg gac tgc caa 2118atg ggc tgg ctg cac tgg gag aga ttc atg tgc aac ctg gac tgc caa 2118

Met Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys GlnMet Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gln

45 50 5545 50 55

gag gaa ccc gac agc tgc atc agc gag aag ctg ttc atg gaa atg gcc 2166gag gaa ccc gac agc tgc atc agc gag aag ctg ttc atg gaa atg gcc 2166

Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met AlaGlu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala

60 65 7060 65 70

gag ctg atg gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac ctg 2214gag ctg atg gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac ctg 2214

Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr LeuGlu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu

75 80 8575 80 85

tgc atc gac gac tgt tgg atg gcc cct cag aga gac tct gag ggc aga 2262tgc atc gac gac tgt tgg atg gcc cct cag aga gac tct gag ggc aga 2262

Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly ArgCys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg

90 95 100 10590 95 100 105

ctg cag gcc gat cct cag aga ttt ccc cac ggc att aga cag ctg gcc 2310ctg cag gcc gat cct cag aga ttt ccc cac ggc att aga cag ctg gcc 2310

Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu AlaLeu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala

110 115 120110 115 120

aac tac gtg cac agc aag ggc ctg aag ctg ggc atc tac gcc gac gtg 2358aac tac gtg cac agc aag ggc ctg aag ctg ggc atc tac gcc gac gtg 2358

Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp ValAsn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val

125 130 135125 130 135

ggc aac aag acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac gat 2406ggc aac aag acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac gat 2406

Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr AspGly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp

140 145 150140 145 150

atc gac gcc cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag ttc 2454atc gac gcc cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag ttc 2454

Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys PheIle Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe

155 160 165155 160 165

gac ggc tgc tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac aag 2502gac ggc tgc tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac aag 2502

Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr LysAsp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys

170 175 180 185170 175 180 185

cac atg tct ctg gcc ctg aat cgg acc ggc aga tcc atc gtg tac agc 2550cac atg tct ctg gcc ctg aat cgg acc ggc aga tcc atc gtg tac agc 2550

His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr SerHis Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser

190 195 200190 195 200

tgc gag tgg ccc ctg tac atg tgg ccc ttc cag aag cct aac tac acc 2598tgc gag tgg ccc ctg tac atg tgg ccc ttc cag aag cct aac tac acc 2598

Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr ThrCys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr

205 210 215205 210 215

gag atc aga cag tac tgc aac cac tgg cgg aac ttc gcc gac atc tgc 2646gag atc aga cag tac tgc aac cac tgg cgg aac ttc gcc gac atc tgc 2646

Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile CysGlu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Cys

220 225 230220 225 230

gat agc tgg aag tcc atc aag agc atc ctg gac tgg acc agc ttc aat 2694gat agc tgg aag tcc atc aag agc atc ctg gac tgg acc agc ttc aat 2694

Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe AsnAsp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn

235 240 245235 240 245

caa gag cgg atc gtg gac gtg gca gga cct ggc gga tgg aac gat cct 2742caa gag cgg atc gtg gac gtg gca gga cct ggc gga tgg aac gat cct 2742

Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp ProGln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro

250 255 260 265250 255 260 265

gac atg ctg gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa gtg 2790gac atg ctg gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa gtg 2790

Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln ValAsp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val

270 275 280270 275 280

acc cag atg gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg agc 2838acc cag atg gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg agc 2838

Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met SerThr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser

285 290 295285 290 295

aac gac ctg aga cac atc agc cct cag gcc aag gct ctg ctg cag gac 2886aac gac ctg aga cac atc agc cct cag gcc aag gct ctg ctg cag gac 2886

Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln AspAsn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp

300 305 310300 305 310

aag gat gtg atc gct atc aac cag gat cct ctg ggc aag cag ggc tac 2934aag gat gtg atc gct atc aac cag gat cct ctg ggc aag cag ggc tac 2934

Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly TyrLys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr

315 320 325315 320 325

cag ctg aga cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg agc 2982cag ctg aga cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg agc 2982

Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu SerGln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser

330 335 340 345330 335 340 345

gga ctg gct tgg gcc gtc gcc atg atc aac cgg caa gag tgc ggc ggc 3030gga ctg gct tgg gcc gtc gcc atg atc aac cgg caa gag tgc ggc ggc 3030

Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Cys Gly GlyGly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Cys Gly Gly

350 355 360350 355 360

ccc aga tcc tac aca atc gcc gtg gcc agt ctc ggc aaa ggc gtg gca 3078ccc aga tcc tac aca atc gcc gtg gcc agt ctc ggc aaa ggc gtg gca 3078

Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val AlaPro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala

365 370 375365 370 375

tgt aat ccc gcc tgc ttc atc aca cag ctg ctg ccc gtg aag aga aag 3126tgt aat ccc gcc tgc ttc atc aca cag ctg ctg ccc gtg aag aga aag 3126

Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg LysCys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys

380 385 390380 385 390

ctg ggc ttt tac gag tgg acc agc aga ctg cgg agc cac atc aat cct 3174ctg ggc ttt tac gag tgg acc agc aga ctg cgg agc cac atc aat cct 3174

Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn ProLeu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro

395 400 405395 400 405

acc ggc aca gtg ctg ctg cag ctg gaa aac acc atg cag atg agc ctg 3222acc ggc aca gtg ctg ctg cag ctg gaa aac acc atg cag atg agc ctg 3222

Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser LeuThr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu

410 415 420 425410 415 420 425

aag gac ctg ctg tga tag aagcttatcg ataatcaacc tctggattac 3270aag gac ctg ctg tga tag aagctttatcg ataatcaacc tctggattac 3270

Lys Asp Leu LeuLys Asp Leu Leu

aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga 3330aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga 3330

tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc 3390tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc 3390

tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa 3450tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa 3450

cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc 3510cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc 3510

acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc 3570acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc 3570

atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc 3630atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc 3630

gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg 3690gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg 3690

attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct 3750attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct 3750

tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg 3810tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg 3810

agtcggatct ccctttgggc cgcctccccg catcgatacc gtctcgaatc aagcggccgc 3870agtcggatct ccctttgggc cgcctccccg catcgatacc gtctcgaatc aagcggccgc 3870

aagcatgctg agctccagat ctggtacctc tagagtcgac ccgggcggcc tcgaggacgg 3930aagcatgctg agctccagat ctggtacctc tagagtcgac ccgggcggcc tcgaggacgg 3930

ggtgaactac gcctgaggat ccgatctttt tccctctgcc aaaaattatg gggacatcat 3990ggtgaactac gcctgaggat ccgatctttt tccctctgcc aaaaattatg gggacatcat 3990

gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt 4050gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt 4050

gtgttggaat tttttgtgtc tctcactcgg aagcaattcg ttgatctgaa tttcgaccac 4110gtgttggaat tttttgtgtc tctcactcgg aagcaattcg ttgatctgaa tttcgaccac 4110

ccataatacc cattaccctg gtagataagt agcatggcgg gttaatcatt aactacaagg 4170ccataatacc cattaccctg gtagataagt agcatggcgg gttaatcatt aactacaagg 4170

aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 4230aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg 4230

ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 4290ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag 4290

cgcgcag 4297cgcgcag 4297

<210> 7<210> 7

<211> 429<211> 429

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 7<400> 7

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 151 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 3020 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 4535 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleArg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 6050 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlySer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 8065 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 9585 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220210 215 220

His Trp Arg Asn Phe Ala Asp Ile Cys Asp Ser Trp Lys Ser Ile LysHis Trp Arg Asn Phe Ala Asp Ile Cys Asp Ser Trp Lys Ser Ile Lys

225 230 235 240225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350340 345 350

Met Ile Asn Arg Gln Glu Cys Gly Gly Pro Arg Ser Tyr Thr Ile AlaMet Ile Asn Arg Gln Glu Cys Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu LeuLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425420 425

<210> 8<210> 8

<211> 3390<211> 3390

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> TBG.PI.hGLAnat.WPRE.bGH<223> TBG.PI.hGLAnat.WPRE.bGH

<220><220>

<221> repeat_region<221> repeat_region

<222> (1)..(168)<222> (1)..(168)

<223> 5' ITR<223> 5' ITR

<220><220>

<221> enhancer<221> enhancer

<222> (211)..(310)<222> (211)..(310)

<223> α mic/bik<223> α mic/bik

<220><220>

<221> enhancer<221> enhancer

<222> (317)..(416)<222> (317)..(416)

<223> α mic/bik<223> α mic/bik

<220><220>

<221> misc_feature<221> misc_feature

<222> (431)..(907)<222> (431)..(907)

<223> TBG启动子<223> TBG promoter

<220><220>

<221> Intron<221> Intron

<222> (939)..(1071)<222> (939)..(1071)

<223> SV40 misc内含子<223> SV40 misc intron

<220><220>

<221> CDS<221> CDS

<222> (1100)..(2392)<222> (1100)..(2392)

<223> hGLA天然<223> hGLA natural

<220><220>

<221> misc_feature<221> misc_feature

<222> (2411)..(2952)<222> (2411)..(2952)

<223> WPRE<223> WPRE

<220><220>

<221> polyA_signal<221> polyA_signal

<222> (2959)..(3173)<222> (2959)..(3173)

<223> BGH pA<223> BGH pA

<220><220>

<221> repeat_region<221> repeat_region

<222> (3223)..(3390)<222> (3223)..(3390)

<223> 3' ITR<223> 3' ITR

<400> 8<400> 8

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240

caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300

caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360

cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420

ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480

tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540

aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600

tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660

taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720

ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780

gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840

taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900

tgagagactg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac 960tgagagactg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac 960

aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 1020aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 1020

gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccagg 1080gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccagg 1080

cggccgcgaa ttcgccacc atg cag ctg agg aac cca gaa cta cat ctg ggc 1132cggccgcgaa ttcgccacc atg cag ctg agg aac cca gaa cta cat ctg ggc 1132

Met Gln Leu Arg Asn Pro Glu Leu His Leu GlyMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly

1 5 101 5 10

tgc gcg ctt gcg ctt cgc ttc ctg gcc ctc gtt tcc tgg gac atc cct 1180tgc gcg ctt gcg ctt cgc ttc ctg gcc ctc gtt tcc tgg gac atc cct 1180

Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp Ile ProCys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro

15 20 2515 20 25

ggg gct aga gca ctg gac aat gga ttg gca agg acg cct acc atg ggc 1228ggg gct aga gca ctg gac aat gga ttg gca agg acg cct acc atg ggc 1228

Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met GlyGly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly

30 35 4030 35 40

tgg ctg cac tgg gag cgc ttc atg tgc aac ctt gac tgc cag gaa gag 1276tgg ctg cac tgg gag cgc ttc atg tgc aac ctt gac tgc cag gaa gag 1276

Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu GluTrp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu

45 50 5545 50 55

cca gat tcc tgc atc agt gag aag ctc ttc atg gag atg gca gag ctc 1324cca gat tcc tgc atc agt gag aag ctc ttc atg gag atg gca gag ctc 1324

Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu LeuPro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu

60 65 70 7560 65 70 75

atg gtc tca gaa ggc tgg aag gat gca ggt tat gag tac ctc tgc att 1372atg gtc tca gaa ggc tgg aag gat gca ggt tat gag tac ctc tgc att 1372

Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys IleMet Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile

80 85 9080 85 90

gat gac tgt tgg atg gct ccc caa aga gat tca gaa ggc aga ctt cag 1420gat gac tgt tgg atg gct ccc caa aga gat tca gaa ggc aga ctt cag 1420

Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu GlnAsp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln

95 100 10595 100 105

gca gac cct cag cgc ttt cct cat ggg att cgc cag cta gct aat tat 1468gca gac cct cag cgc ttt cct cat ggg att cgc cag cta gct aat tat 1468

Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn TyrAla Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr

110 115 120110 115 120

gtt cac agc aaa gga ctg aag cta ggg att tat gca gat gtt gga aat 1516gtt cac agc aaa gga ctg aag cta ggg att tat gca gat gtt gga aat 1516

Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly AsnVal His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn

125 130 135125 130 135

aaa acc tgc gca ggc ttc cct ggg agt ttt gga tac tac gac att gat 1564aaa acc tgc gca ggc ttc cct ggg agt ttt gga tac tac gac att gat 1564

Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile AspLys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp

140 145 150 155140 145 150 155

gcc cag acc ttt gct gac tgg gga gta gat ctg cta aaa ttt gat ggt 1612gcc cag acc ttt gct gac tgg gga gta gat ctg cta aaa ttt gat ggt 1612

Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp GlyAla Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly

160 165 170160 165 170

tgt tac tgt gac agt ttg gaa aat ttg gca gat ggt tat aag cac atg 1660tgt tac tgt gac agt ttg gaa aat ttg gca gat ggt tat aag cac atg 1660

Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His MetCys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met

175 180 185175 180 185

tcc ttg gcc ctg aat agg act ggc aga agc att gtg tac tcc tgt gag 1708tcc ttg gcc ctg aat agg act ggc aga agc att gtg tac tcc tgt gag 1708

Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys GluSer Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu

190 195 200190 195 200

tgg cct ctt tat atg tgg ccc ttt caa aag ccc aat tat aca gaa atc 1756tgg cct ctt tat atg tgg ccc ttt caa aag ccc aat tat aca gaa atc 1756

Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu IleTrp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile

205 210 215205 210 215

cga cag tac tgc aat cac tgg cga aat ttt gct gac att gat gat tcc 1804cga cag tac tgc aat cac tgg cga aat ttt gct gac att gat gat tcc 1804

Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp SerArg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser

220 225 230 235220 225 230 235

tgg aaa agt ata aag agt atc ttg gac tgg aca tct ttt aac cag gag 1852tgg aaa agt ata aag agt atc ttg gac tgg aca tct ttt aac cag gag 1852

Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln GluTrp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu

240 245 250240 245 250

aga att gtt gat gtt gct gga cca ggg ggt tgg aat gac cca gat atg 1900aga att gtt gat gtt gct gga cca ggg ggt tgg aat gac cca gat atg 1900

Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp MetArg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met

255 260 265255 260 265

tta gtg att ggc aac ttt ggc ctc agc tgg aat cag caa gta act cag 1948tta gtg att ggc aac ttt ggc ctc agc tgg aat cag caa gta act cag 1948

Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr GlnLeu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln

270 275 280270 275 280

atg gcc ctc tgg gct atc atg gct gct cct tta ttc atg tct aat gac 1996atg gcc ctc tgg gct atc atg gct gct cct tta ttc atg tct aat gac 1996

Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn AspMet Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp

285 290 295285 290 295

ctc cga cac atc agc cct caa gcc aaa gct ctc ctt cag gat aag gac 2044ctc cga cac atc agc cct caa gcc aaa gct ctc ctt cag gat aag gac 2044

Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys AspLeu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp

300 305 310 315300 305 310 315

gta att gcc atc aat cag gac ccc ttg ggc aag caa ggg tac cag ctt 2092gta att gcc atc aat cag gac ccc ttg ggc aag caa ggg tac cag ctt 2092

Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln LeuVal Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu

320 325 330320 325 330

aga cag gga gac aac ttt gaa gtg tgg gaa cga cct ctc tca ggc tta 2140aga cag gga gac aac ttt gaa gtg tgg gaa cga cct ctc tca ggc tta 2140

Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly LeuArg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu

335 340 345335 340 345

gcc tgg gct gta gct atg ata aac cgg cag gag att ggt gga cct cgc 2188gcc tgg gct gta gct atg ata aac cgg cag gag att ggt gga cct cgc 2188

Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly Gly Pro ArgAla Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg

350 355 360350 355 360

tct tat acc atc gca gtt gct tcc ctg ggt aaa gga gtg gcc tgt aat 2236tct tat acc atc gca gtt gct tcc ctg ggt aaa gga gtg gcc tgt aat 2236

Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys AsnSer Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn

365 370 375365 370 375

cct gcc tgc ttc atc aca cag ctc ctc cct gtg aaa agg aag cta ggg 2284cct gcc tgc ttc atc aca cag ctc ctc cct gtg aaa agg aag cta ggg 2284

Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu GlyPro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly

380 385 390 395380 385 390 395

ttc tat gaa tgg act tca agg tta aga agt cac ata aat ccc aca ggc 2332ttc tat gaa tgg act tca agg tta aga agt cac ata aat ccc aca ggc 2332

Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr GlyPhe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly

400 405 410400 405 410

act gtt ttg ctt cag cta gaa aat aca atg cag atg tca tta aaa gac 2380act gtt ttg ctt cag cta gaa aat aca atg cag atg tca tta aaa gac 2380

Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys AspThr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp

415 420 425415 420 425

tta ctt taa tga tgtacaaagc ttggatccaa tcaacctctg gattacaaaa 2432tta ctt taa tga tgtacaaagc ttggatccaa tcaacctctg gattacaaaa 2432

Leu LeuLeu Leu

tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 2492tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 2492

ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2552ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2552

tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2612tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2612

gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2672gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2672

gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 2732gtcagctcctttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 2732

ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 2792ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 2792

tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 2852tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 2852

tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 2912tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 2912

gcggcctgct gccggctctg cggcctcttc cgcgtcttcg agatctgcct cgactgtgcc 2972gcggcctgct gccggctctg cggcctcttc cgcgtcttcg agatctgcct cgactgtgcc 2972

ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 3032ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 3032

tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 3092tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 3092

gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 3152gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 3152

caatagcagg catgctgggg actcgagtta agggcgaatt cccgataagg atcttcctag 3212caatagcagg catgctgggg actcgagtta agggcgaatt cccgataagg atcttcctag 3212

agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag 3272agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag 3272

tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 3332tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 3332

aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcag 3390aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcag 3390

<210> 9<210> 9

<211> 429<211> 429

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 9<400> 9

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 151 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 3020 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 4535 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleArg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 6050 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlySer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 8065 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 9585 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile LysHis Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile AlaMet Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu LeuLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425420 425

<210> 10<210> 10

<211> 4294<211> 4294

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> CB7.CI.hGLAnat.WPRE.RBG<223> CB7.CI.hGLAnat.WPRE.RBG

<220><220>

<221> repeat_region<221> repeat_region

<222> (1)..(130)<222> (1)..(130)

<223> 5' ITR<223> 5' ITR

<220><220>

<221> misc_feature<221> misc_feature

<222> (198)..(579)<222> (198)..(579)

<223> CMV IE启动子<223> CMV IE promoter

<220><220>

<221> promoter<221> promoter

<222> (582)..(863)<222> (582)..(863)

<223> CB启动子<223> CB promoter

<220><220>

<221> TATA_signal<221> TATA_signal

<222> (836)..(839)<222> (836)..(839)

<223> TATA<223> TATA

<220><220>

<221> Intron<221> Intron

<222> (956)..(1928)<222> (956)..(1928)

<223> 鸡β-肌动蛋白内含子<223> Chicken β-actin intron

<220><220>

<221> CDS<221> CDS

<222> (1950)..(3242)<222> (1950)..(3242)

<223> hGLA天然<223> hGLA natural

<220><220>

<221> misc_feature<221> misc_feature

<222> (3317)..(3905)<222> (3317)..(3905)

<223> WPRE<223> WPRE

<220><220>

<221> polyA_signal<221> polyA_signal

<222> (3950)..(4076)<222> (3950)..(4076)

<223> 兔珠蛋白poly A<223> Rabbit globin poly A

<220><220>

<221> repeat_region<221> repeat_region

<222> (4165)..(4294)<222> (4165)..(4294)

<223> 3' ITR<223> 3' ITR

<400> 10<400> 10

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960

cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440

ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1800tctccagcct cggggctgtc cgcgggggga cggctgcctt cggggggggac ggggcagggc 1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920

tggcaaagaa tagcttcgaa ttcgccacc atg cag ctg agg aac cca gaa cta 1973tggcaaagaa tagcttcgaa ttcgccacc atg cag ctg agg aac cca gaa cta 1973

Met Gln Leu Arg Asn Pro Glu LeuMet Gln Leu Arg Asn Pro Glu Leu

1 51 5

cat ctg ggc tgc gcg ctt gcg ctt cgc ttc ctg gcc ctc gtt tcc tgg 2021cat ctg ggc tgc gcg ctt gcg ctt cgc ttc ctg gcc ctc gtt tcc tgg 2021

His Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser TrpHis Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp

10 15 2010 15 20

gac atc cct ggg gct aga gca ctg gac aat gga ttg gca agg acg cct 2069gac atc cct ggg gct aga gca ctg gac aat gga ttg gca agg acg cct 2069

Asp Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr ProAsp Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro

25 30 35 4025 30 35 40

acc atg ggc tgg ctg cac tgg gag cgc ttc atg tgc aac ctt gac tgc 2117acc atg ggc tgg ctg cac tgg gag cgc ttc atg tgc aac ctt gac tgc 2117

Thr Met Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp CysThr Met Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys

45 50 5545 50 55

cag gaa gag cca gat tcc tgc atc agt gag aag ctc ttc atg gag atg 2165cag gaa gag cca gat tcc tgc atc agt gag aag ctc ttc atg gag atg 2165

Gln Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu MetGln Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met

60 65 7060 65 70

gca gag ctc atg gtc tca gaa ggc tgg aag gat gca ggt tat gag tac 2213gca gag ctc atg gtc tca gaa ggc tgg aag gat gca ggt tat gag tac 2213

Ala Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu TyrAla Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr

75 80 8575 80 85

ctc tgc att gat gac tgt tgg atg gct ccc caa aga gat tca gaa ggc 2261ctc tgc att gat gac tgt tgg atg gct ccc caa aga gat tca gaa ggc 2261

Leu Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu GlyLeu Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly

90 95 10090 95 100

aga ctt cag gca gac cct cag cgc ttt cct cat ggg att cgc cag cta 2309aga ctt cag gca gac cct cag cgc ttt cct cat ggg att cgc cag cta 2309

Arg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln LeuArg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu

105 110 115 120105 110 115 120

gct aat tat gtt cac agc aaa gga ctg aag cta ggg att tat gca gat 2357gct aat tat gtt cac agc aaa gga ctg aag cta ggg att tat gca gat 2357

Ala Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala AspAla Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp

125 130 135125 130 135

gtt gga aat aaa acc tgc gca ggc ttc cct ggg agt ttt gga tac tac 2405gtt gga aat aaa acc tgc gca ggc ttc cct ggg agt ttt gga tac tac 2405

Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr TyrVal Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr

140 145 150140 145 150

gac att gat gcc cag acc ttt gct gac tgg gga gta gat ctg cta aaa 2453gac att gat gcc cag acc ttt gct gac tgg gga gta gat ctg cta aaa 2453

Asp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu LysAsp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys

155 160 165155 160 165

ttt gat ggt tgt tac tgt gac agt ttg gaa aat ttg gca gat ggt tat 2501ttt gat ggt tgt tac tgt gac agt ttg gaa aat ttg gca gat ggt tat 2501

Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly TyrPhe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr

170 175 180170 175 180

aag cac atg tcc ttg gcc ctg aat agg act ggc aga agc att gtg tac 2549aag cac atg tcc ttg gcc ctg aat agg act ggc aga agc att gtg tac 2549

Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val TyrLys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr

185 190 195 200185 190 195 200

tcc tgt gag tgg cct ctt tat atg tgg ccc ttt caa aag ccc aat tat 2597tcc tgt gag tgg cct ctt tat atg tgg ccc ttt caa aag ccc aat tat 2597

Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn TyrSer Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr

205 210 215205 210 215

aca gaa atc cga cag tac tgc aat cac tgg cga aat ttt gct gac att 2645aca gaa atc cga cag tac tgc aat cac tgg cga aat ttt gct gac att 2645

Thr Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp IleThr Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile

220 225 230220 225 230

gat gat tcc tgg aaa agt ata aag agt atc ttg gac tgg aca tct ttt 2693gat gat tcc tgg aaa agt ata aag agt atc ttg gac tgg aca tct ttt 2693

Asp Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser PheAsp Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe

235 240 245235 240 245

aac cag gag aga att gtt gat gtt gct gga cca ggg ggt tgg aat gac 2741aac cag gag aga att gtt gat gtt gct gga cca ggg ggt tgg aat gac 2741

Asn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn AspAsn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp

250 255 260250 255 260

cca gat atg tta gtg att ggc aac ttt ggc ctc agc tgg aat cag caa 2789cca gat atg tta gtg att ggc aac ttt ggc ctc agc tgg aat cag caa 2789

Pro Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln GlnPro Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln

265 270 275 280265 270 275 280

gta act cag atg gcc ctc tgg gct atc atg gct gct cct tta ttc atg 2837gta act cag atg gcc ctc tgg gct atc atg gct gct cct tta ttc atg 2837

Val Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe MetVal Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met

285 290 295285 290 295

tct aat gac ctc cga cac atc agc cct caa gcc aaa gct ctc ctt cag 2885tct aat gac ctc cga cac atc agc cct caa gcc aaa gct ctc ctt cag 2885

Ser Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu GlnSer Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln

300 305 310300 305 310

gat aag gac gta att gcc atc aat cag gac ccc ttg ggc aag caa ggg 2933gat aag gac gta att gcc atc aat cag gac ccc ttg ggc aag caa ggg 2933

Asp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln GlyAsp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly

315 320 325315 320 325

tac cag ctt aga cag gga gac aac ttt gaa gtg tgg gaa cga cct ctc 2981tac cag ctt aga cag gga gac aac ttt gaa gtg tgg gaa cga cct ctc 2981

Tyr Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro LeuTyr Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu

330 335 340330 335 340

tca ggc tta gcc tgg gct gta gct atg ata aac cgg cag gag att ggt 3029tca ggc tta gcc tgg gct gta gct atg ata aac cgg cag gag att ggt 3029

Ser Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile GlySer Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly

345 350 355 360345 350 355 360

gga cct cgc tct tat acc atc gca gtt gct tcc ctg ggt aaa gga gtg 3077gga cct cgc tct tat acc atc gca gtt gct tcc ctg ggt aaa gga gtg 3077

Gly Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly ValGly Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val

365 370 375365 370 375

gcc tgt aat cct gcc tgc ttc atc aca cag ctc ctc cct gtg aaa agg 3125gcc tgt aat cct gcc tgc ttc atc aca cag ctc ctc cct gtg aaa agg 3125

Ala Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys ArgAla Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg

380 385 390380 385 390

aag cta ggg ttc tat gaa tgg act tca agg tta aga agt cac ata aat 3173aag cta ggg ttc tat gaa tgg act tca agg tta aga agt cac ata aat 3173

Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile AsnLys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn

395 400 405395 400 405

ccc aca ggc act gtt ttg ctt cag cta gaa aat aca atg cag atg tca 3221ccc aca ggc act gtt ttg ctt cag cta gaa aat aca atg cag atg tca 3221

Pro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met SerPro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser

410 415 420410 415 420

tta aaa gac tta ctt taa tga tgtacaagta aagatctgcg gccgcgtggt 3272tta aaa gac tta ctt taa tga tgtacaagta aagatctgcg gccgcgtggt 3272

Leu Lys Asp Leu LeuLeu Lys Asp Leu Leu

425425

acctctagag tcgacccggg cggcctcgaa tcaagcttat cgataatcaa cctctggatt 3332acctctagag tcgacccggg cggcctcgaa tcaagcttat cgataatcaa cctctggatt 3332

acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 3392acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 3392

gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 3452gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 3452

cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 3512cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 3512

aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 3572aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 3572

ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 3632ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 3632

tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3692tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3692

ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 3752ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 3752

ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3812ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3812

cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 3872cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 3872

cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtctcgag gacggggtga 3932cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtctcgag gacggggtga 3932

actacgcctg aggatccgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc 3992actacgcctg aggatccgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc 3992

cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt 4052cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt 4052

ggaatttttt gtgtctctca ctcggaagca attcgttgat ctgaatttcg accacccata 4112ggaatttttt gtgtctctca ctcggaagca attcgttgat ctgaatttcg accacccata 4112

atacccatta ccctggtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc 4172atacccatta ccctggtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc 4172

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 4232ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 4232

ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4292ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4292

ag 4294ag 4294

<210> 11<210> 11

<211> 429<211> 429

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 11<400> 11

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 151 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 3020 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 4535 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleArg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 6050 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlySer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 8065 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 9585 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile LysHis Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile AlaMet Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu LeuLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425420 425

<210> 12<210> 12

<211> 3390<211> 3390

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> TBG.PI.hGLAco.WPRE.bGH<223> TBG.PI.hGLAco.WPRE.bGH

<220><220>

<221> repeat_region<221> repeat_region

<222> (1)..(168)<222> (1)..(168)

<223> 5' ITR<223> 5' ITR

<220><220>

<221> enhancer<221> enhancer

<222> (211)..(310)<222> (211)..(310)

<220><220>

<221> enhancer<221> enhancer

<222> (317)..(416)<222> (317)..(416)

<220><220>

<221> Intron<221> Intron

<222> (939)..(1071)<222> (939)..(1071)

<223> SV40 misc内含子<223> SV40 misc intron

<220><220>

<221> CDS<221> CDS

<222> (1100)..(2392)<222> (1100)..(2392)

<223> hGLAco<223> hGLAco

<220><220>

<221> misc_feature<221> misc_feature

<222> (2411)..(2952)<222> (2411)..(2952)

<223> WPRE<223> WPRE

<220><220>

<221> polyA_signal<221> polyA_signal

<222> (2959)..(3173)<222> (2959)..(3173)

<223> BGH pA<223> BGH pA

<220><220>

<221> repeat_region<221> repeat_region

<222> (3223)..(3390)<222> (3223)..(3390)

<223> 3' ITR<223> 3' ITR

<400> 12<400> 12

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240

caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300

caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360

cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420

ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480

tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540

aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600

tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660

taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720

ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780

gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840

taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900

tgagagactg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac 960tgagagactg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac 960

aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 1020aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 1020

gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccagg 1080gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccagg 1080

cggccgcgaa ttcgccacc atg cag ctg aga aat ccc gag ctg cac ctg ggc 1132cggccgcgaa ttcgccacc atg cag ctg aga aat ccc gag ctg cac ctg ggc 1132

Met Gln Leu Arg Asn Pro Glu Leu His Leu GlyMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly

1 5 101 5 10

tgt gcc ctg gct ctg aga ttt ctg gcc ctg gtg tct tgg gac atc cct 1180tgt gcc ctg gct ctg aga ttt ctg gcc ctg gtg tct tgg gac atc cct 1180

Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp Ile ProCys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro

15 20 2515 20 25

ggc gct aga gcc ctg gat aac ggc ctg gcc aga aca cct aca atg ggc 1228ggc gct aga gcc ctg gat aac ggc ctg gcc aga aca cct aca atg ggc 1228

Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met GlyGly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly

30 35 4030 35 40

tgg ctg cac tgg gag aga ttc atg tgc aac ctg gac tgc caa gag gaa 1276tgg ctg cac tgg gag aga ttc atg tgc aac ctg gac tgc caa gag gaa 1276

Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu GluTrp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu

45 50 5545 50 55

ccc gac agc tgc atc agc gag aag ctg ttc atg gaa atg gcc gag ctg 1324ccc gac agc tgc atc agc gag aag ctg ttc atg gaa atg gcc gag ctg 1324

Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu LeuPro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu

60 65 70 7560 65 70 75

atg gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac ctg tgc atc 1372atg gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac ctg tgc atc 1372

Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys IleMet Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile

80 85 9080 85 90

gac gac tgt tgg atg gcc cct cag aga gac tct gag ggc aga ctg cag 1420gac gac tgt tgg atg gcc cct cag aga gac tct gag ggc aga ctg cag 1420

Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu GlnAsp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln

95 100 10595 100 105

gcc gat cct cag aga ttt ccc cac ggc att aga cag ctg gcc aac tac 1468gcc gat cct cag aga ttt ccc cac ggc att aga cag ctg gcc aac tac 1468

Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn TyrAla Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr

110 115 120110 115 120

gtg cac agc aag ggc ctg aag ctg ggc atc tac gcc gac gtg ggc aac 1516gtg cac agc aag ggc ctg aag ctg ggc atc tac gcc gac gtg ggc aac 1516

Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly AsnVal His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn

125 130 135125 130 135

aag acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac gat atc gac 1564aag acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac gat atc gac 1564

Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile AspLys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp

140 145 150 155140 145 150 155

gcc cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag ttc gac ggc 1612gcc cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag ttc gac ggc 1612

Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp GlyAla Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly

160 165 170160 165 170

tgc tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac aag cac atg 1660tgc tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac aag cac atg 1660

Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His MetCys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met

175 180 185175 180 185

tca ctg gcc ctg aat cgg acc ggc cgc agc atc gtg tac tct tgc gag 1708tca ctg gcc ctg aat cgg acc ggc cgc agc atc gtg tac tct tgc gag 1708

Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys GluSer Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu

190 195 200190 195 200

tgg ccc ctg tat atg tgg ccc ttc cag aag cct aac tac acc gag atc 1756tgg ccc ctg tat atg tgg ccc ttc cag aag cct aac tac acc gag atc 1756

Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu IleTrp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile

205 210 215205 210 215

aga cag tac tgc aac cac tgg cgg aac ttc gcc gac atc gac gat agc 1804aga cag tac tgc aac cac tgg cgg aac ttc gcc gac atc gac gat agc 1804

Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp SerArg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser

220 225 230 235220 225 230 235

tgg aag tcc atc aag agc atc ctg gac tgg acc agc ttc aat caa gag 1852tgg aag tcc atc aag agc atc ctg gac tgg acc agc ttc aat caa gag 1852

Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln GluTrp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu

240 245 250240 245 250

cgg atc gtg gac gtg gca gga cct ggc gga tgg aac gat cct gac atg 1900cgg atc gtg gac gtg gca gga cct ggc gga tgg aac gat cct gac atg 1900

Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp MetArg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met

255 260 265255 260 265

ctg gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa gtg acc cag 1948ctg gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa gtg acc cag 1948

Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr GlnLeu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln

270 275 280270 275 280

atg gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg agc aac gac 1996atg gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg agc aac gac 1996

Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn AspMet Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp

285 290 295285 290 295

ctg aga cac atc agc cct cag gcc aag gct ctg ctg cag gac aag gat 2044ctg aga cac atc agc cct cag gcc aag gct ctg ctg cag gac aag gat 2044

Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys AspLeu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp

300 305 310 315300 305 310 315

gtg atc gct atc aac cag gat cct ctg ggc aag cag ggc tac cag ctg 2092gtg atc gct atc aac cag gat cct ctg ggc aag cag ggc tac cag ctg 2092

Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln LeuVal Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu

320 325 330320 325 330

aga cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg agc gga ctg 2140aga cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg agc gga ctg 2140

Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly LeuArg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu

335 340 345335 340 345

gct tgg gcc gtc gcc atg atc aac aga caa gag atc ggc gga ccc cgg 2188gct tgg gcc gtc gcc atg atc aac aga caa gag atc ggc gga ccc cgg 2188

Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly Gly Pro ArgAla Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg

350 355 360350 355 360

tcc tac aca att gcc gtg gct tct ctc ggc aaa ggc gtg gcc tgt aat 2236tcc tac aca att gcc gtg gct tct ctc ggc aaa ggc gtg gcc tgt aat 2236

Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys AsnSer Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn

365 370 375365 370 375

ccc gcc tgc ttt atc aca cag ctg ctg ccc gtg aag aga aag ctg ggc 2284ccc gcc tgc ttt atc aca cag ctg ctg ccc gtg aag aga aag ctg ggc 2284

Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu GlyPro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly

380 385 390 395380 385 390 395

ttt tac gag tgg acc agc aga ctg cgg agc cac atc aat cct acc ggc 2332ttt tac gag tgg acc agc aga ctg cgg agc cac atc aat cct acc ggc 2332

Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr GlyPhe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly

400 405 410400 405 410

aca gtg ctg ctg cag ctg gaa aac aca atg cag atg agc ctg aag gac 2380aca gtg ctg ctg cag ctg gaa aac aca atg cag atg agc ctg aag gac 2380

Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys AspThr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp

415 420 425415 420 425

ctg ctg tga tga tgtacaaagc ttggatccaa tcaacctctg gattacaaaa 2432ctg ctg tga tga tgtacaaagc ttggatccaa tcaacctctg gattacaaaa 2432

Leu LeuLeu Leu

tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 2492tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 2492

ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2552ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2552

tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2612tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2612

gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2672gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2672

gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 2732gtcagctcctttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 2732

ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 2792ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 2792

tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 2852tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 2852

tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 2912tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 2912

gcggcctgct gccggctctg cggcctcttc cgcgtcttcg agatctgcct cgactgtgcc 2972gcggcctgct gccggctctg cggcctcttc cgcgtcttcg agatctgcct cgactgtgcc 2972

ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 3032ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 3032

tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 3092tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 3092

gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 3152gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 3152

caatagcagg catgctgggg actcgagtta agggcgaatt cccgataagg atcttcctag 3212caatagcagg catgctgggg actcgagtta agggcgaatt cccgataagg atcttcctag 3212

agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag 3272agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag 3272

tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 3332tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 3332

aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcag 3390aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcag 3390

<210> 13<210> 13

<211> 429<211> 429

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 13<400> 13

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 151 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 3020 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 4535 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleArg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 6050 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlySer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 8065 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 9585 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile LysHis Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile AlaMet Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu LeuLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425420 425

<210> 14<210> 14

<211> 4294<211> 4294

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> CB7.CI.hGLAco.WPRE.RBG<223> CB7.CI.hGLAco.WPRE.RBG

<220><220>

<221> repeat_region<221> repeat_region

<222> (1)..(130)<222> (1)..(130)

<223> 5' ITR<223> 5' ITR

<220><220>

<221> repeat_region<221> repeat_region

<222> (198)..(579)<222> (198)..(579)

<223> CMV IE启动子<223> CMV IE promoter

<220><220>

<221> promoter<221> promoter

<222> (582)..(863)<222> (582)..(863)

<223> CB启动子<223> CB promoter

<220><220>

<221> TATA_signal<221> TATA_signal

<222> (582)..(863)<222> (582)..(863)

<223> TATA<223> TATA

<220><220>

<221> Intron<221> Intron

<222> (956)..(1928)<222> (956)..(1928)

<223> 鸡β-肌动蛋白内含子<223> Chicken β-actin intron

<220><220>

<221> CDS<221> CDS

<222> (1950)..(3242)<222> (1950)..(3242)

<223> hGLAco<223> hGLAco

<220><220>

<221> misc_feature<221> misc_feature

<222> (3317)..(3905)<222> (3317)..(3905)

<223> WPRE<223> WPRE

<220><220>

<221> polyA_signal<221> polyA_signal

<222> (3950)..(4076)<222> (3950)..(4076)

<223> 兔珠蛋白poly A<223> Rabbit globin poly A

<220><220>

<221> repeat_region<221> repeat_region

<222> (4165)..(4294)<222> (4165)..(4294)

<223> 3' ITR<223> 3' ITR

<400> 14<400> 14

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960

cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440

ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1800tctccagcct cggggctgtc cgcgggggga cggctgcctt cggggggggac ggggcagggc 1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920

tggcaaagaa tagcttcgaa ttcgccacc atg cag ctg aga aat ccc gag ctg 1973tggcaaagaa tagcttcgaa ttcgccacc atg cag ctg aga aat ccc gag ctg 1973

Met Gln Leu Arg Asn Pro Glu LeuMet Gln Leu Arg Asn Pro Glu Leu

1 51 5

cac ctg ggc tgt gcc ctg gct ctg aga ttt ctg gcc ctg gtg tct tgg 2021cac ctg ggc tgt gcc ctg gct ctg aga ttt ctg gcc ctg gtg tct tgg 2021

His Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser TrpHis Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp

10 15 2010 15 20

gac atc cct ggc gct aga gcc ctg gat aac ggc ctg gcc aga aca cct 2069gac atc cct ggc gct aga gcc ctg gat aac ggc ctg gcc aga aca cct 2069

Asp Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr ProAsp Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro

25 30 35 4025 30 35 40

aca atg ggc tgg ctg cac tgg gag aga ttc atg tgc aac ctg gac tgc 2117aca atg ggc tgg ctg cac tgg gag aga ttc atg tgc aac ctg gac tgc 2117

Thr Met Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp CysThr Met Gly Trp Leu His Trp Glu Arg Phe Met Cys Asn Leu Asp Cys

45 50 5545 50 55

caa gag gaa ccc gac agc tgc atc agc gag aag ctg ttc atg gaa atg 2165caa gag gaa ccc gac agc tgc atc agc gag aag ctg ttc atg gaa atg 2165

Gln Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu MetGln Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met

60 65 7060 65 70

gcc gag ctg atg gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac 2213gcc gag ctg atg gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac 2213

Ala Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu TyrAla Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr

75 80 8575 80 85

ctg tgc atc gac gac tgt tgg atg gcc cct cag aga gac tct gag ggc 2261ctg tgc atc gac gac tgt tgg atg gcc cct cag aga gac tct gag ggc 2261

Leu Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu GlyLeu Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly

90 95 10090 95 100

aga ctg cag gcc gat cct cag aga ttt ccc cac ggc att aga cag ctg 2309aga ctg cag gcc gat cct cag aga ttt ccc cac ggc att aga cag ctg 2309

Arg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln LeuArg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu

105 110 115 120105 110 115 120

gcc aac tac gtg cac agc aag ggc ctg aag ctg ggc atc tac gcc gac 2357gcc aac tac gtg cac agc aag ggc ctg aag ctg ggc atc tac gcc gac 2357

Ala Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala AspAla Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp

125 130 135125 130 135

gtg ggc aac aag acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac 2405gtg ggc aac aag acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac 2405

Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr TyrVal Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr

140 145 150140 145 150

gat atc gac gcc cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag 2453gat atc gac gcc cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag 2453

Asp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu LysAsp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys

155 160 165155 160 165

ttc gac ggc tgc tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac 2501ttc gac ggc tgc tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac 2501

Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly TyrPhe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr

170 175 180170 175 180

aag cac atg tca ctg gcc ctg aat cgg acc ggc cgc agc atc gtg tac 2549aag cac atg tca ctg gcc ctg aat cgg acc ggc cgc agc atc gtg tac 2549

Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val TyrLys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr

185 190 195 200185 190 195 200

tct tgc gag tgg ccc ctg tat atg tgg ccc ttc cag aag cct aac tac 2597tct tgc gag tgg ccc ctg tat atg tgg ccc ttc cag aag cct aac tac 2597

Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn TyrSer Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr

205 210 215205 210 215

acc gag atc aga cag tac tgc aac cac tgg cgg aac ttc gcc gac atc 2645acc gag atc aga cag tac tgc aac cac tgg cgg aac ttc gcc gac atc 2645

Thr Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp IleThr Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile

220 225 230220 225 230

gac gat agc tgg aag tcc atc aag agc atc ctg gac tgg acc agc ttc 2693gac gat agc tgg aag tcc atc aag agc atc ctg gac tgg acc agc ttc 2693

Asp Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser PheAsp Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe

235 240 245235 240 245

aat caa gag cgg atc gtg gac gtg gca gga cct ggc gga tgg aac gat 2741aat caa gag cgg atc gtg gac gtg gca gga cct ggc gga tgg aac gat 2741

Asn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn AspAsn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp

250 255 260250 255 260

cct gac atg ctg gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa 2789cct gac atg ctg gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa 2789

Pro Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln GlnPro Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln

265 270 275 280265 270 275 280

gtg acc cag atg gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg 2837gtg acc cag atg gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg 2837

Val Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe MetVal Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met

285 290 295285 290 295

agc aac gac ctg aga cac atc agc cct cag gcc aag gct ctg ctg cag 2885agc aac gac ctg aga cac atc agc cct cag gcc aag gct ctg ctg cag 2885

Ser Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu GlnSer Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln

300 305 310300 305 310

gac aag gat gtg atc gct atc aac cag gat cct ctg ggc aag cag ggc 2933gac aag gat gtg atc gct atc aac cag gat cct ctg ggc aag cag ggc 2933

Asp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln GlyAsp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly

315 320 325315 320 325

tac cag ctg aga cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg 2981tac cag ctg aga cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg 2981

Tyr Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro LeuTyr Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu

330 335 340330 335 340

agc gga ctg gct tgg gcc gtc gcc atg atc aac aga caa gag atc ggc 3029agc gga ctg gct tgg gcc gtc gcc atg atc aac aga caa gag atc ggc 3029

Ser Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile GlySer Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Gly

345 350 355 360345 350 355 360

gga ccc cgg tcc tac aca att gcc gtg gct tct ctc ggc aaa ggc gtg 3077gga ccc cgg tcc tac aca att gcc gtg gct tct ctc ggc aaa ggc gtg 3077

Gly Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly ValGly Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val

365 370 375365 370 375

gcc tgt aat ccc gcc tgc ttt atc aca cag ctg ctg ccc gtg aag aga 3125gcc tgt aat ccc gcc tgc ttt atc aca cag ctg ctg ccc gtg aag aga 3125

Ala Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys ArgAla Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg

380 385 390380 385 390

aag ctg ggc ttt tac gag tgg acc agc aga ctg cgg agc cac atc aat 3173aag ctg ggc ttt tac gag tgg acc agc aga ctg cgg agc cac atc aat 3173

Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile AsnLys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn

395 400 405395 400 405

cct acc ggc aca gtg ctg ctg cag ctg gaa aac aca atg cag atg agc 3221cct acc ggc aca gtg ctg ctg cag ctg gaa aac aca atg cag atg agc 3221

Pro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met SerPro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser

410 415 420410 415 420

ctg aag gac ctg ctg tga tga tgtacaagta aagatctgcg gccgcgtggt 3272ctg aag gac ctg ctg tga tga tgtacaagta aagatctgcg gccgcgtggt 3272

Leu Lys Asp Leu LeuLeu Lys Asp Leu Leu

425425

acctctagag tcgacccggg cggcctcgaa tcaagcttat cgataatcaa cctctggatt 3332acctctagag tcgacccggg cggcctcgaa tcaagcttat cgataatcaa cctctggatt 3332

acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 3392acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 3392

gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 3452gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 3452

cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 3512cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 3512

aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 3572aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 3572

ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 3632ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 3632

tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3692tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3692

ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 3752ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 3752

ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3812ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3812

cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 3872cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 3872

cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtctcgag gacggggtga 3932cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtctcgag gacggggtga 3932

actacgcctg aggatccgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc 3992actacgcctg aggatccgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc 3992

cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt 4052cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt 4052

ggaatttttt gtgtctctca ctcggaagca attcgttgat ctgaatttcg accacccata 4112ggaatttttt gtgtctctca ctcggaagca attcgttgat ctgaatttcg accacccata 4112

atacccatta ccctggtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc 4172atacccatta ccctggtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc 4172

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 4232ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 4232

ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4292ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4292

ag 4294ag 4294

<210> 15<210> 15

<211> 429<211> 429

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 15<400> 15

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 151 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 3020 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 4535 40 45

Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleArg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 6050 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlySer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 8065 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 9585 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile LysHis Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile AlaMet Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu LeuLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425420 425

<210> 16<210> 16

<211> 3378<211> 3378

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> TBG.PI.hGLAco(M51C_G360C).WPRE.bGH<223> TBG.PI.hGLAco(M51C_G360C).WPRE.bGH

<220><220>

<221> repeat_region<221> repeat_region

<222> (1)..(168)<222> (1)..(168)

<223> 5' ITR<223> 5' ITR

<220><220>

<221> enhancer<221> enhancer

<222> (211)..(310)<222> (211)..(310)

<223> α mic/bik<223> α mic/bik

<220><220>

<221> enhancer<221> enhancer

<222> (317)..(416)<222> (317)..(416)

<223> α mic/bik<223> α mic/bik

<220><220>

<221> misc_feature<221> misc_feature

<222> (431)..(907)<222> (431)..(907)

<223> TBG启动子<223> TBG promoter

<220><220>

<221> Intron<221> Intron

<222> (939)..(1071)<222> (939)..(1071)

<223> SV40 misc内含子<223> SV40 misc intron

<220><220>

<221> CDS<221> CDS

<222> (1094)..(2386)<222> (1094)..(2386)

<223> hGLAco.M51C.G360C<223> hGLAco.M51C.G360C

<220><220>

<221> misc_feature<221> misc_feature

<222> (2399)..(2940)<222> (2399)..(2940)

<223> WPRE<223> WPRE

<220><220>

<221> polyA_signal<221> polyA_signal

<222> (2947)..(3161)<222> (2947)..(3161)

<223> BGH pA<223> BGH pA

<220><220>

<221> repeat_region<221> repeat_region

<222> (3211)..(3378)<222> (3211)..(3378)

<223> 3' ITR<223> 3' ITR

<400> 16<400> 16

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240

caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300

caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360

cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420

ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480

tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540

aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600

tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660

taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720

ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780

gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840

taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900

tgagagactg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac 960tgagagactg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac 960

aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 1020aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct 1020

gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccagg 1080gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccagg 1080

cggccgcgcc acc atg caa ctg aga aat cct gaa ctg cac ctg ggc tgc 1129cggccgcgcc acc atg caa ctg aga aat cct gaa ctg cac ctg ggc tgc 1129

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly CysMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys

1 5 101 5 10

gcc ctg gct ctg aga ttt ctg gct ctg gtg tcc tgg gac atc cct ggc 1177gcc ctg gct ctg aga ttt ctg gct ctg gtg tcc tgg gac atc cct ggc 1177

Ala Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro GlyAla Leu Ala Leu Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly

15 20 2515 20 25

gct aga gcc ctg gat aac ggc ctg gcc aga aca cct aca atg ggc tgg 1225gct aga gcc ctg gat aac ggc ctg gcc aga aca cct aca atg ggc tgg 1225

Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly TrpAla Arg Ala Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp

30 35 4030 35 40

ctg cac tgg gag aga ttc tgc tgc aac ctg gac tgc caa gag gaa ccc 1273ctg cac tgg gag aga ttc tgc tgc aac ctg gac tgc caa gag gaa ccc 1273

Leu His Trp Glu Arg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu ProLeu His Trp Glu Arg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu Pro

45 50 55 6045 50 55 60

gac agc tgc atc agc gag aag ctg ttc atg gaa atg gcc gag ctg atg 1321gac agc tgc atc agc gag aag ctg ttc atg gaa atg gcc gag ctg atg 1321

Asp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu MetAsp Ser Cys Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met

65 70 7565 70 75

gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac ctg tgc atc gac 1369gtg tcc gaa ggc tgg aag gac gcc ggc tac gag tac ctg tgc atc gac 1369

Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile AspVal Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp

80 85 9080 85 90

gac tgt tgg atg gcc cct cag aga gac tct gag ggc aga ctg cag gcc 1417gac tgt tgg atg gcc cct cag aga gac tct gag ggc aga ctg cag gcc 1417

Asp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln AlaAsp Cys Trp Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala

95 100 10595 100 105

gat cct cag aga ttt ccc cac ggc att aga cag ctg gcc aac tac gtg 1465gat cct cag aga ttt ccc cac ggc att aga cag ctg gcc aac tac gtg 1465

Asp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr ValAsp Pro Gln Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val

110 115 120110 115 120

cac agc aag ggc ctg aag ctg ggc atc tac gcc gac gtg ggc aac aag 1513cac agc aag ggc ctg aag ctg ggc atc tac gcc gac gtg ggc aac aag 1513

His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn LysHis Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys

125 130 135 140125 130 135 140

acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac gat atc gac gcc 1561acc tgt gcc ggc ttt cct ggc agc ttc ggc tac tac gat atc gac gcc 1561

Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp AlaThr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala

145 150 155145 150 155

cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag ttc gac ggc tgc 1609cag acc ttc gcc gat tgg gga gtc gat ctg ctg aag ttc gac ggc tgc 1609

Gln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly CysGln Thr Phe Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys

160 165 170160 165 170

tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac aag cac atg tct 1657tac tgc gac agc ctg gaa aat ctg gcc gac ggc tac aag cac atg tct 1657

Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met SerTyr Cys Asp Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser

175 180 185175 180 185

ctg gcc ctg aat cgg acc ggc aga tcc atc gtg tac agc tgc gag tgg 1705ctg gcc ctg aat cgg acc ggc aga tcc atc gtg tac agc tgc gag tgg 1705

Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu TrpLeu Ala Leu Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp

190 195 200190 195 200

ccc ctg tac atg tgg ccc ttc cag aag cct aac tac acc gag atc aga 1753ccc ctg tac atg tgg ccc ttc cag aag cct aac tac acc gag atc aga 1753

Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile ArgPro Leu Tyr Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg

205 210 215 220205 210 215 220

cag tac tgc aac cac tgg cgg aac ttc gcc gac atc gac gat agc tgg 1801cag tac tgc aac cac tgg cgg aac ttc gcc gac atc gac gat agc tgg 1801

Gln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser TrpGln Tyr Cys Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp

225 230 235225 230 235

aag tcc atc aag agc atc ctg gac tgg acc agc ttc aat caa gag cgg 1849aag tcc atc aag agc atc ctg gac tgg acc agc ttc aat caa gag cgg 1849

Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu ArgLys Ser Ile Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg

240 245 250240 245 250

atc gtg gac gtg gca gga cct ggc gga tgg aac gat cct gac atg ctg 1897atc gtg gac gtg gca gga cct ggc gga tgg aac gat cct gac atg ctg 1897

Ile Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met LeuIle Val Asp Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu

255 260 265255 260 265

gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa gtg acc cag atg 1945gtc atc ggc aac ttc ggc ctg agc tgg aac cag caa gtg acc cag atg 1945

Val Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln MetVal Ile Gly Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met

270 275 280270 275 280

gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg agc aac gac ctg 1993gcc ctg tgg gcc att atg gcc gct cct ctg ttc atg agc aac gac ctg 1993

Ala Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp LeuAla Leu Trp Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu

285 290 295 300285 290 295 300

aga cac atc agc cct cag gcc aag gct ctg ctg cag gac aag gat gtg 2041aga cac atc agc cct cag gcc aag gct ctg ctg cag gac aag gat gtg 2041

Arg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp ValArg His Ile Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val

305 310 315305 310 315

atc gct atc aac cag gat cct ctg ggc aag cag ggc tac cag ctg aga 2089atc gct atc aac cag gat cct ctg ggc aag cag ggc tac cag ctg aga 2089

Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu ArgIle Ala Ile Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg

320 325 330320 325 330

cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg agc gga ctg gct 2137cag ggc gac aat ttc gaa gtg tgg gaa aga ccc ctg agc gga ctg gct 2137

Gln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu AlaGln Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala

335 340 345335 340 345

tgg gcc gtc gcc atg atc aac cgg caa gag att tgc ggc ccc aga tcc 2185tgg gcc gtc gcc atg atc aac cgg caa gag att tgc ggc ccc aga tcc 2185

Trp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg SerTrp Ala Val Ala Met Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser

350 355 360350 355 360

tac aca atc gcc gtg gcc agt ctc ggc aaa ggc gtg gca tgt aat ccc 2233tac aca atc gcc gtg gcc agt ctc ggc aaa ggc gtg gca tgt aat ccc 2233

Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn ProTyr Thr Ile Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro

365 370 375 380365 370 375 380

gcc tgc ttc atc aca cag ctg ctg ccc gtg aag aga aag ctg ggc ttt 2281gcc tgc ttc atc aca cag ctg ctg ccc gtg aag aga aag ctg ggc ttt 2281

Ala Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly PheAla Cys Phe Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe

385 390 395385 390 395

tac gag tgg acc agc aga ctg cgg agc cac atc aat cct acc ggc aca 2329tac gag tgg acc agc aga ctg cgg agc cac atc aat cct acc ggc aca 2329

Tyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly ThrTyr Glu Trp Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr

400 405 410400 405 410

gtg ctg ctg cag ctg gaa aac acc atg cag atg agc ctg aag gac ctg 2377gtg ctg ctg cag ctg gaa aac acc atg cag atg agc ctg aag gac ctg 2377

Val Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp LeuVal Leu Leu Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu

415 420 425415 420 425

ctg tga tag aagcttggat ccaatcaacc tctggattac aaaatttgtg 2426ctg tga tag aagcttggat ccaatcaacc tctggattac aaaatttgtg 2426

LeuLeu

aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 2486aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 2486

taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 2546taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 2546

aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 2606aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 2606

tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 2666tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 2666

tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 2726tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 2726

gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 2786gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 2786

cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg 2846cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg 2846

ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 2906ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 2906

tgctgccggc tctgcggcct cttccgcgtc ttcgagatct gcctcgactg tgccttctag 2966tgctgccggc tctgcggcct cttccgcgtc ttcgagatct gcctcgactg tgccttctag 2966

ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 3026ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 3026

tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 3086tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 3086

ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 3146ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 3146

caggcatgct ggggactcga gttaagggcg aattcccgat aaggatcttc ctagagcatg 3206caggcatgct ggggactcga gttaagggcg aattcccgat aaggatcttc ctagagcatg 3206

gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg 3266gctacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg 3266

agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg 3326agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg 3326

cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc ag 3378cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc ag 3378

<210> 17<210> 17

<211> 429<211> 429

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 17<400> 17

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 151 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 3020 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 4535 40 45

Arg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleArg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 6050 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlySer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 8065 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 9585 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile LysHis Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350340 345 350

Met Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser Tyr Thr Ile AlaMet Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu LeuLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425420 425

<210> 18<210> 18

<211> 4240<211> 4240

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> CB7.CI.hGLAco(M51C_G360C).WPRE.RBG<223> CB7.CI.hGLAco(M51C_G360C).WPRE.RBG

<220><220>

<221> repeat_region<221> repeat_region

<222> (1)..(130)<222> (1)..(130)

<223> 5' ITR<223> 5' ITR

<220><220>

<221> misc_feature<221> misc_feature

<222> (198)..(579)<222> (198)..(579)

<223> CMV IE启动子<223> CMV IE promoter

<220><220>

<221> promoter<221> promoter

<222> (582)..(863)<222> (582)..(863)

<223> CB启动子<223> CB promoter

<220><220>

<221> TATA_signal<221> TATA_signal

<222> (836)..(839)<222> (836)..(839)

<223> TATA<223> TATA

<220><220>

<221> Intron<221> Intron

<222> (956)..(1928)<222> (956)..(1928)

<223> 鸡β-肌动蛋白内含子<223> Chicken β-actin intron

<220><220>

<221> CDS<221> CDS

<222> (1958)..(3250)<222> (1958)..(3250)

<223> hGLAco.M51C.G360C<223> hGLAco.M51C.G360C

<220><220>

<221> misc_feature<221> misc_feature

<222> (2363)..(2382)<222> (2363)..(2382)

<223> P18<223> P18

<220><220>

<221> misc_feature<221> misc_feature

<222> (3263)..(3851)<222> (3263)..(3851)

<223> WPRE<223> WPRE

<220><220>

<221> misc_feature<221> misc_feature

<222> (3619)..(3638)<222> (3619)..(3638)

<223> P19<223> P19

<220><220>

<221> polyA_signal<221> polyA_signal

<222> (3896)..(4022)<222> (3896)..(4022)

<223> 兔珠蛋白poly A<223> Rabbit globin poly A

<220><220>

<221> repeat_region<221> repeat_region

<222> (4111)..(4240)<222> (4111)..(4240)

<223> 3' ITR<223> 3' ITR

<400> 18<400> 18

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180

atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 420

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540

tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600

gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660

tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720

ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780

gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840

aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc 900

cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 960

cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 1020

ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 1080

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 1140

gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 1200

gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 1260

caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 1320

cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1380

tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1440

ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1500

cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1560

atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1620

atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1680

caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1740

tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1800tctccagcct cggggctgtc cgcgggggga cggctgcctt cggggggggac ggggcagggc 1800

ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1860

cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1920

tggcaaagaa tagcttcgaa ttcgcggccg cgccacc atg caa ctg aga aat cct 1975tggcaaagaa tagcttcgaa ttcgcggccg cgccacc atg caa ctg aga aat cct 1975

Met Gln Leu Arg Asn ProMet Gln Leu Arg Asn Pro

1 51 5

gaa ctg cac ctg ggc tgc gcc ctg gct ctg aga ttt ctg gct ctg gtg 2023gaa ctg cac ctg ggc tgc gcc ctg gct ctg aga ttt ctg gct ctg gtg 2023

Glu Leu His Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu ValGlu Leu His Leu Gly Cys Ala Leu Ala Leu Arg Phe Leu Ala Leu Val

10 15 2010 15 20

tcc tgg gac atc cct ggc gct aga gcc ctg gat aac ggc ctg gcc aga 2071tcc tgg gac atc cct ggc gct aga gcc ctg gat aac ggc ctg gcc aga 2071

Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala ArgSer Trp Asp Ile Pro Gly Ala Arg Ala Leu Asp Asn Gly Leu Ala Arg

25 30 3525 30 35

aca cct aca atg ggc tgg ctg cac tgg gag aga ttc tgc tgc aac ctg 2119aca cct aca atg ggc tgg ctg cac tgg gag aga ttc tgc tgc aac ctg 2119

Thr Pro Thr Met Gly Trp Leu His Trp Glu Arg Phe Cys Cys Asn LeuThr Pro Thr Met Gly Trp Leu His Trp Glu Arg Phe Cys Cys Asn Leu

40 45 5040 45 50

gac tgc caa gag gaa ccc gac agc tgc atc agc gag aag ctg ttc atg 2167gac tgc caa gag gaa ccc gac agc tgc atc agc gag aag ctg ttc atg 2167

Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe MetAsp Cys Gln Glu Glu Pro Asp Ser Cys Ile Ser Glu Lys Leu Phe Met

55 60 65 7055 60 65 70

gaa atg gcc gag ctg atg gtg tcc gaa ggc tgg aag gac gcc ggc tac 2215gaa atg gcc gag ctg atg gtg tcc gaa ggc tgg aag gac gcc ggc tac 2215

Glu Met Ala Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly TyrGlu Met Ala Glu Leu Met Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr

75 80 8575 80 85

gag tac ctg tgc atc gac gac tgt tgg atg gcc cct cag aga gac tct 2263gag tac ctg tgc atc gac gac tgt tgg atg gcc cct cag aga gac tct 2263

Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp SerGlu Tyr Leu Cys Ile Asp Asp Cys Trp Met Ala Pro Gln Arg Asp Ser

90 95 10090 95 100

gag ggc aga ctg cag gcc gat cct cag aga ttt ccc cac ggc att aga 2311gag ggc aga ctg cag gcc gat cct cag aga ttt ccc cac ggc att aga 2311

Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile ArgGlu Gly Arg Leu Gln Ala Asp Pro Gln Arg Phe Pro His Gly Ile Arg

105 110 115105 110 115

cag ctg gcc aac tac gtg cac agc aag ggc ctg aag ctg ggc atc tac 2359cag ctg gcc aac tac gtg cac agc aag ggc ctg aag ctg ggc atc tac 2359

Gln Leu Ala Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile TyrGln Leu Ala Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr

120 125 130120 125 130

gcc gac gtg ggc aac aag acc tgt gcc ggc ttt cct ggc agc ttc ggc 2407gcc gac gtg ggc aac aag acc tgt gcc ggc ttt cct ggc agc ttc ggc 2407

Ala Asp Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe GlyAla Asp Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly

135 140 145 150135 140 145 150

tac tac gat atc gac gcc cag acc ttc gcc gat tgg gga gtc gat ctg 2455tac tac gat atc gac gcc cag acc ttc gcc gat tgg gga gtc gat ctg 2455

Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp LeuTyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala Asp Trp Gly Val Asp Leu

155 160 165155 160 165

ctg aag ttc gac ggc tgc tac tgc gac agc ctg gaa aat ctg gcc gac 2503ctg aag ttc gac ggc tgc tac tgc gac agc ctg gaa aat ctg gcc gac 2503

Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala AspLeu Lys Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala Asp

170 175 180170 175 180

ggc tac aag cac atg tct ctg gcc ctg aat cgg acc ggc aga tcc atc 2551ggc tac aag cac atg tct ctg gcc ctg aat cgg acc ggc aga tcc atc 2551

Gly Tyr Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser IleGly Tyr Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg Ser Ile

185 190 195185 190 195

gtg tac agc tgc gag tgg ccc ctg tac atg tgg ccc ttc cag aag cct 2599gtg tac agc tgc gag tgg ccc ctg tac atg tgg ccc ttc cag aag cct 2599

Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys ProVal Tyr Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe Gln Lys Pro

200 205 210200 205 210

aac tac acc gag atc aga cag tac tgc aac cac tgg cgg aac ttc gcc 2647aac tac acc gag atc aga cag tac tgc aac cac tgg cgg aac ttc gcc 2647

Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe AlaAsn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn His Trp Arg Asn Phe Ala

215 220 225 230215 220 225 230

gac atc gac gat agc tgg aag tcc atc aag agc atc ctg gac tgg acc 2695gac atc gac gat agc tgg aag tcc atc aag agc atc ctg gac tgg acc 2695

Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp ThrAsp Ile Asp Asp Ser Trp Lys Ser Ile Lys Ser Ile Leu Asp Trp Thr

235 240 245235 240 245

agc ttc aat caa gag cgg atc gtg gac gtg gca gga cct ggc gga tgg 2743agc ttc aat caa gag cgg atc gtg gac gtg gca gga cct ggc gga tgg 2743

Ser Phe Asn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly TrpSer Phe Asn Gln Glu Arg Ile Val Asp Val Ala Gly Pro Gly Gly Trp

250 255 260250 255 260

aac gat cct gac atg ctg gtc atc ggc aac ttc ggc ctg agc tgg aac 2791aac gat cct gac atg ctg gtc atc ggc aac ttc ggc ctg agc tgg aac 2791

Asn Asp Pro Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp AsnAsn Asp Pro Asp Met Leu Val Ile Gly Asn Phe Gly Leu Ser Trp Asn

265 270 275265 270 275

cag caa gtg acc cag atg gcc ctg tgg gcc att atg gcc gct cct ctg 2839cag caa gtg acc cag atg gcc ctg tgg gcc att atg gcc gct cct ctg 2839

Gln Gln Val Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro LeuGln Gln Val Thr Gln Met Ala Leu Trp Ala Ile Met Ala Ala Pro Leu

280 285 290280 285 290

ttc atg agc aac gac ctg aga cac atc agc cct cag gcc aag gct ctg 2887ttc atg agc aac gac ctg aga cac atc agc cct cag gcc aag gct ctg 2887

Phe Met Ser Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala LeuPhe Met Ser Asn Asp Leu Arg His Ile Ser Pro Gln Ala Lys Ala Leu

295 300 305 310295 300 305 310

ctg cag gac aag gat gtg atc gct atc aac cag gat cct ctg ggc aag 2935ctg cag gac aag gat gtg atc gct atc aac cag gat cct ctg ggc aag 2935

Leu Gln Asp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly LysLeu Gln Asp Lys Asp Val Ile Ala Ile Asn Gln Asp Pro Leu Gly Lys

315 320 325315 320 325

cag ggc tac cag ctg aga cag ggc gac aat ttc gaa gtg tgg gaa aga 2983cag ggc tac cag ctg aga cag ggc gac aat ttc gaa gtg tgg gaa aga 2983

Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu ArgGln Gly Tyr Gln Leu Arg Gln Gly Asp Asn Phe Glu Val Trp Glu Arg

330 335 340330 335 340

ccc ctg agc gga ctg gct tgg gcc gtc gcc atg atc aac cgg caa gag 3031ccc ctg agc gga ctg gct tgg gcc gtc gcc atg atc aac cgg caa gag 3031

Pro Leu Ser Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln GluPro Leu Ser Gly Leu Ala Trp Ala Val Ala Met Ile Asn Arg Gln Glu

345 350 355345 350 355

att tgc ggc ccc aga tcc tac aca atc gcc gtg gcc agt ctc ggc aaa 3079att tgc ggc ccc aga tcc tac aca atc gcc gtg gcc agt ctc ggc aaa 3079

Ile Cys Gly Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly LysIle Cys Gly Pro Arg Ser Tyr Thr Ile Ala Val Ala Ser Leu Gly Lys

360 365 370360 365 370

ggc gtg gca tgt aat ccc gcc tgc ttc atc aca cag ctg ctg ccc gtg 3127ggc gtg gca tgt aat ccc gcc tgc ttc atc aca cag ctg ctg ccc gtg 3127

Gly Val Ala Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro ValGly Val Ala Cys Asn Pro Ala Cys Phe Ile Thr Gln Leu Leu Pro Val

375 380 385 390375 380 385 390

aag aga aag ctg ggc ttt tac gag tgg acc agc aga ctg cgg agc cac 3175aag aga aag ctg ggc ttt tac gag tgg acc agc aga ctg cgg agc cac 3175

Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser HisLys Arg Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His

395 400 405395 400 405

atc aat cct acc ggc aca gtg ctg ctg cag ctg gaa aac acc atg cag 3223atc aat cct acc ggc aca gtg ctg ctg cag ctg gaa aac acc atg cag 3223

Ile Asn Pro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met GlnIle Asn Pro Thr Gly Thr Val Leu Leu Gln Leu Glu Asn Thr Met Gln

410 415 420410 415 420

atg agc ctg aag gac ctg ctg tga tag aagcttatcg ataatcaacc 3270atg agc ctg aag gac ctg ctg tga tag aagctttcg ataatcaacc 3270

Met Ser Leu Lys Asp Leu LeuMet Ser Leu Lys Asp Leu Leu

425425

tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 3330tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 3330

gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 3390gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 3390

cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 3450cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 3450

tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3510tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3510

cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3570cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3570

ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3630ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3630

tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 3690tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 3690

tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 3750tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 3750

ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 3810ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 3810

ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtctcgagga 3870ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtctcgagga 3870

cggggtgaac tacgcctgag gatccgatct ttttccctct gccaaaaatt atggggacat 3930cggggtgaac tacgcctgag gatccgatct ttttccctct gccaaaaatt atggggacat 3930

catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat 3990catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat 3990

agtgtgttgg aattttttgt gtctctcact cggaagcaat tcgttgatct gaatttcgac 4050agtgtgttgg aattttttgt gtctctcact cggaagcaat tcgttgatct gaatttcgac 4050

cacccataat acccattacc ctggtagata agtagcatgg cgggttaatc attaactaca 4110cacccataat acccattacc ctggtagata agtagcatgg cgggttaatc attaactaca 4110

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 4170aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 4170

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 4230ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 4230

gagcgcgcag 4240gagcgcgcag 4240

<210> 19<210> 19

<211> 429<211> 429

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 19<400> 19

Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala LeuMet Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu

1 5 10 151 5 10 15

Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala LeuArg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu

20 25 3020 25 30

Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp GluAsp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu

35 40 4535 40 45

Arg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys IleArg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile

50 55 6050 55 60

Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu GlySer Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly

65 70 75 8065 70 75 80

Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp MetTrp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met

85 90 9585 90 95

Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln ArgAla Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg

100 105 110100 105 110

Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys GlyPhe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly

115 120 125115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala GlyLeu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe AlaPhe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp SerAsp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu AsnLeu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr MetArg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys AsnTrp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile LysHis Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp ValSer Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly AsnAla Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp AlaPhe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile SerIle Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile AsnPro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp AsnGln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val AlaPhe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350340 345 350

Met Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser Tyr Thr Ile AlaMet Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe IleVal Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp ThrThr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu GlnSer Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu LeuLeu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425420 425

<210> 20<210> 20

<211> 2211<211> 2211

<212> DNA<212> DNA

<213> 腺相关病毒人68<213> Adeno-associated virus human 68

<220><220>

<221> CDS<221> CDS

<222> (1)..(2211)<222> (1)..(2211)

<400> 20<400> 20

atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctc agt 48atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctc agt 48

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu SerMet Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 151 5 10 15

gaa ggc att cgc gag tgg tgg gct ttg aaa cct gga gcc cct caa ccc 96gaa ggc att cgc gag tgg tgg gct ttg aaa cct gga gcc cct caa ccc 96

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln ProGlu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 3020 25 30

aag gca aat caa caa cat caa gac aac gct cgg ggt ctt gtg ctt ccg 144aag gca aat caa caa cat caa gac aac gct cgg ggt ctt gtg ctt ccg 144

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu ProLys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 4535 40 45

ggt tac aaa tac ctt gga ccc ggc aac gga ctc gac aag ggg gag ccg 192ggt tac aaa tac ctt gga ccc ggc aac gga ctc gac aag ggg gag ccg 192

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu ProGly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 6050 55 60

gtc aac gaa gca gac gcg gcg gcc ctc gag cac gac aag gcc tac gac 240gtc aac gaa gca gac gcg gcg gcc ctc gag cac gac aag gcc tac gac 240

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr AspVal Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 8065 70 75 80

cag cag ctc aag gcc gga gac aac ccg tac ctc aag tac aac cac gcc 288cag cag ctc aag gcc gga gac aac ccg tac ctc aag tac aac cac gcc 288

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His AlaGln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 9585 90 95

gac gcc gag ttc cag gag cgg ctc aaa gaa gat acg tct ttt ggg ggc 336gac gcc gag ttc cag gag cgg ctc aaa gaa gat acg tct ttt ggg ggc 336

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly GlyAsp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110100 105 110

aac ctc ggg cga gca gtc ttc cag gcc aaa aag agg ctt ctt gaa cct 384aac ctc ggg cga gca gtc ttc cag gcc aaa aag agg ctt ctt gaa cct 384

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu ProAsn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125115 120 125

ctt ggt ctg gtt gag gaa gcg gct aag acg gct cct gga aag aag agg 432ctt ggt ctg gtt gag gaa gcg gct aag acg gct cct gga aag aag agg 432

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys ArgLeu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140130 135 140

cct gta gag cag tct cct cag gaa ccg gac tcc tcc gtg ggt att ggc 480cct gta gag cag tct cct cag gaa ccg gac tcc tcc gtg ggt att ggc 480

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Val Gly Ile GlyPro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Val Gly Ile Gly

145 150 155 160145 150 155 160

aaa tcg ggt gca cag ccc gct aaa aag aga ctc aat ttc ggt cag act 528aaa tcg ggt gca cag ccc gct aaa aag aga ctc aat ttc ggt cag act 528

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln ThrLys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175165 170 175

ggc gac aca gag tca gtc ccc gac cct caa cca atc gga gaa cct ccc 576ggc gac aca gag tca gtc ccc gac cct caa cca atc gga gaa cct ccc 576

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro ProGly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190180 185 190

gca gcc ccc tca ggt gtg gga tct ctt aca atg gct tca ggt ggt ggc 624gca gcc ccc tca ggt gtg gga tct ctt aca atg gct tca ggt ggt ggc 624

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly GlyAla Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205195 200 205

gca cca gtg gca gac aat aac gaa ggt gcc gat gga gtg ggt agt tcc 672gca cca gtg gca gac aat aac gaa ggt gcc gat gga gtg ggt agt tcc 672

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser SerAla Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220210 215 220

tcg gga aat tgg cat tgc gat tcc caa tgg ctg ggg gac aga gtc atc 720tcg gga aat tgg cat tgc gat tcc caa tgg ctg ggg gac aga gtc atc 720

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val IleSer Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240225 230 235 240

acc acc agc acc cga acc tgg gcc ctg ccc acc tac aac aat cac ctc 768acc acc agc acc cga acc tgg gcc ctg ccc acc tac aac aat cac ctc 768

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His LeuThr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255245 250 255

tac aag caa atc tcc aac agc aca tct gga gga tct tca aat gac aac 816tac aag caa atc tcc aac agc aca tct gga gga tct tca aat gac aac 816

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp AsnTyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270260 265 270

gcc tac ttc ggc tac agc acc ccc tgg ggg tat ttt gac ttc aac aga 864gcc tac ttc ggc tac agc acc ccc tgg ggg tat ttt gac ttc aac aga 864

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn ArgAla Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285275 280 285

ttc cac tgc cac ttc tca cca cgt gac tgg caa aga ctc atc aac aac 912ttc cac tgc cac ttc tca cca cgt gac tgg caa aga ctc atc aac aac 912

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn AsnPhe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300290 295 300

aac tgg gga ttc cgg cct aag cga ctc aac ttc aag ctc ttc aac att 960aac tgg gga ttc cgg cct aag cga ctc aac ttc aag ctc ttc aac att 960

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn IleAsn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320305 310 315 320

cag gtc aaa gag gtt acg gac aac aat gga gtc aag acc atc gct aat 1008cag gtc aaa gag gtt acg gac aac aat gga gtc aag acc atc gct aat 1008

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala AsnGln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335325 330 335

aac ctt acc agc acg gtc cag gtc ttc acg gac tca gac tat cag ctc 1056aac ctt acc agc acg gtc cag gtc ttc acg gac tca gac tat cag ctc 1056

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln LeuAsn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350340 345 350

ccg tac gtg ctc ggg tcg gct cac gag ggc tgc ctc ccg ccg ttc cca 1104ccg tac gtg ctc ggg tcg gct cac gag ggc tgc ctc ccg ccg ttc cca 1104

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe ProPro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365355 360 365

gcg gac gtt ttc atg att cct cag tac ggg tat cta acg ctt aat gat 1152gcg gac gtt ttc atg att cct cag tac ggg tat cta acg ctt aat gat 1152

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn AspAla Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380370 375 380

gga agc caa gcc gtg ggt cgt tcg tcc ttt tac tgc ctg gaa tat ttc 1200gga agc caa gcc gtg ggt cgt tcg tcc ttt tac tgc ctg gaa tat ttc 1200

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr PheGly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400385 390 395 400

ccg tcg caa atg cta aga acg ggt aac aac ttc cag ttc agc tac gag 1248ccg tcg caa atg cta aga acg ggt aac aac ttc cag ttc agc tac gag 1248

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr GluPro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415405 410 415

ttt gag aac gta cct ttc cat agc agc tat gct cac agc caa agc ctg 1296ttt gag aac gta cct ttc cat agc agc tat gct cac agc caa agc ctg 1296

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser LeuPhe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430420 425 430

gac cga ctc atg aat cca ctc atc gac caa tac ttg tac tat ctc tca 1344gac cga ctc atg aat cca ctc atc gac caa tac ttg tac tat ctc tca 1344

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu SerAsp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445435 440 445

aag act att aac ggt tct gga cag aat caa caa acg cta aaa ttc agt 1392aag act att aac ggt tct gga cag aat caa caa acg cta aaa ttc agt 1392

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe SerLys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460450 455 460

gtg gcc gga ccc agc aac atg gct gtc cag gga aga aac tac ata cct 1440gtg gcc gga ccc agc aac atg gct gtc cag gga aga aac tac ata cct 1440

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile ProVal Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480465 470 475 480

gga ccc agc tac cga caa caa cgt gtc tca acc act gtg act caa aac 1488gga ccc agc tac cga caa caa cgt gtc tca acc act gtg act caa aac 1488

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln AsnGly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495485 490 495

aac aac agc gaa ttt gct tgg cct gga gct tct tct tgg gct ctc aat 1536aac aac agc gaa ttt gct tgg cct gga gct tct tct tgg gct ctc aat 1536

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu AsnAsn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510500 505 510

gga cgt aat agc ttg atg aat cct gga cct gct atg gcc agc cac aaa 1584gga cgt aat agc ttg atg aat cct gga cct gct atg gcc agc cac aaa 1584

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His LysGly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525515 520 525

gaa gga gag gac cgt ttc ttt cct ttg tct gga tct tta att ttt ggc 1632gaa gga gag gac cgt ttc ttt cct ttg tct gga tct tta att ttt ggc 1632

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe GlyGlu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540530 535 540

aaa caa gga act gga aga gac aac gtg gat gcg gac aaa gtc atg ata 1680aaa caa gga act gga aga gac aac gtg gat gcg gac aaa gtc atg ata 1680

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met IleLys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560545 550 555 560

acc aac gaa gaa gaa att aaa act acc aac cca gta gca acg gag tcc 1728acc aac gaa gaa gaa att aaa act acc aac cca gta gca acg gag tcc 1728

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu SerThr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575565 570 575

tat gga caa gtg gcc aca aac cac cag agt gcc caa gca cag gcg cag 1776tat gga caa gtg gcc aca aac cac cag agt gcc caa gca cag gcg cag 1776

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala GlnTyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590580 585 590

acc ggc tgg gtt caa aac caa gga ata ctt ccg ggt atg gtt tgg cag 1824acc ggc tgg gtt caa aac caa gga ata ctt ccg ggt atg gtt tgg cag 1824

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp GlnThr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605595 600 605

gac aga gat gtg tac ctg caa gga ccc att tgg gcc aaa att cct cac 1872gac aga gat gtg tac ctg caa gga ccc att tgg gcc aaa att cct cac 1872

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro HisAsp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620610 615 620

acg gac ggc aac ttt cac cct tct ccg ctg atg gga ggg ttt gga atg 1920acg gac ggc aac ttt cac cct tct ccg ctg atg gga ggg ttt gga atg 1920

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly MetThr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640625 630 635 640

aag cac ccg cct cct cag atc ctc atc aaa aac aca cct gta cct gcg 1968aag cac ccg cct cct cag atc ctc atc aaa aac aca cct gta cct gcg 1968

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro AlaLys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655645 650 655

gat cct cca acg gct ttc aac aag gac aag ctg aac tct ttc atc acc 2016gat cct cca acg gct ttc aac aag gac aag ctg aac tct ttc atc acc 2016

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile ThrAsp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670660 665 670

cag tat tct act ggc caa gtc agc gtg gag att gag tgg gag ctg cag 2064cag tat tct act ggc caa gtc agc gtg gag att gag tgg gag ctg cag 2064

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu GlnGln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685675 680 685

aag gaa aac agc aag cgc tgg aac ccg gag atc cag tac act tcc aac 2112aag gaa aac agc aag cgc tgg aac ccg gag atc cag tac act tcc aac 2112

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser AsnLys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700690 695 700

tat tac aag tct aat aat gtt gaa ttt gct gtt aat act gaa ggt gtt 2160tat tac aag tct aat aat gtt gaa ttt gct gtt aat act gaa ggt gtt 2160

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly ValTyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720705 710 715 720

tat tct gaa ccc cgc ccc att ggc acc aga tac ctg act cgt aat ctg 2208tat tct gaa ccc cgc ccc att ggc acc aga tac ctg act cgt aat ctg 2208

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn LeuTyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735725 730 735

taa 2211taa 2211

<210> 21<210> 21

<211> 736<211> 736

<212> PRT<212> PRT

<213> 腺相关病毒人68<213> Adeno-associated virus human 68

<400> 21<400> 21

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu SerMet Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 151 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln ProGlu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 3020 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu ProLys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 4535 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu ProGly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 6050 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr AspVal Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 8065 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His AlaGln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 9585 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly GlyAsp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu ProAsn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys ArgLeu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Val Gly Ile GlyPro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Val Gly Ile Gly

145 150 155 160145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln ThrLys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro ProGly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly GlyAla Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser SerAla Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val IleSer Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His LeuThr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp AsnTyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn ArgAla Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn AsnPhe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn IleAsn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala AsnGln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln LeuAsn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe ProPro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn AspAla Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr PheGly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr GluPro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser LeuPhe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu SerAsp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe SerLys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile ProVal Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln AsnGly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu AsnAsn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His LysGly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe GlyGlu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met IleLys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu SerThr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala GlnTyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp GlnThr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro HisAsp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly MetThr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro AlaLys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile ThrAsp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu GlnGln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser AsnLys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly ValTyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn LeuTyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735725 730 735

<210> 22<210> 22

<211> 2208<211> 2208

<212> DNA<212> DNA

<213> 腺相关病毒9<213> Adeno-associated virus 9

<220><220>

<221> CDS<221> CDS

<222> (1)..(2208)<222> (1)..(2208)

<223> AAV9 VP1衣壳<223> AAV9 VP1 capsid

<400> 22<400> 22

atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctt agt 48atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctt agt 48

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu SerMet Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 151 5 10 15

gaa gga att cgc gag tgg tgg gct ttg aaa cct gga gcc cct caa ccc 96gaa gga att cgc gag tgg tgg gct ttg aaa cct gga gcc cct caa ccc 96

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln ProGlu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 3020 25 30

aag gca aat caa caa cat caa gac aac gct cga ggt ctt gtg ctt ccg 144aag gca aat caa caa cat caa gac aac gct cga ggt ctt gtg ctt ccg 144

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu ProLys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 4535 40 45

ggt tac aaa tac ctt gga ccc ggc aac gga ctc gac aag ggg gag ccg 192ggt tac aaa tac ctt gga ccc ggc aac gga ctc gac aag ggg gag ccg 192

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu ProGly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 6050 55 60

gtc aac gca gca gac gcg gcg gcc ctc gag cac gac aag gcc tac gac 240gtc aac gca gca gac gcg gcg gcc ctc gag cac gac aag gcc tac gac 240

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr AspVal Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 8065 70 75 80

cag cag ctc aag gcc gga gac aac ccg tac ctc aag tac aac cac gcc 288cag cag ctc aag gcc gga gac aac ccg tac ctc aag tac aac cac gcc 288

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His AlaGln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 9585 90 95

gac gcc gag ttc cag gag cgg ctc aaa gaa gat acg tct ttt ggg ggc 336gac gcc gag ttc cag gag cgg ctc aaa gaa gat acg tct ttt ggg ggc 336

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly GlyAsp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110100 105 110

aac ctc ggg cga gca gtc ttc cag gcc aaa aag agg ctt ctt gaa cct 384aac ctc ggg cga gca gtc ttc cag gcc aaa aag agg ctt ctt gaa cct 384

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu ProAsn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125115 120 125

ctt ggt ctg gtt gag gaa gcg gct aag acg gct cct gga aag aag agg 432ctt ggt ctg gtt gag gaa gcg gct aag acg gct cct gga aag aag agg 432

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys ArgLeu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140130 135 140

cct gta gag cag tct cct cag gaa ccg gac tcc tcc gcg ggt att ggc 480cct gta gag cag tct cct cag gaa ccg gac tcc tcc gcg ggt att ggc 480

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile GlyPro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160145 150 155 160

aaa tcg ggt gca cag ccc gct aaa aag aga ctc aat ttc ggt cag act 528aaa tcg ggt gca cag ccc gct aaa aag aga ctc aat ttc ggt cag act 528

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln ThrLys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175165 170 175

ggc gac aca gag tca gtc cca gac cct caa cca atc gga gaa cct ccc 576ggc gac aca gag tca gtc cca gac cct caa cca atc gga gaa cct ccc 576

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro ProGly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190180 185 190

gca gcc ccc tca ggt gtg gga tct ctt aca atg gct tca ggt ggt ggc 624gca gcc ccc tca ggt gtg gga tct ctt aca atg gct tca ggt ggt ggc 624

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly GlyAla Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205195 200 205

gca cca gtg gca gac aat aac gaa ggt gcc gat gga gtg ggt agt tcc 672gca cca gtg gca gac aat aac gaa ggt gcc gat gga gtg ggt agt tcc 672

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser SerAla Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220210 215 220

tcg gga aat tgg cat tgc gat tcc caa tgg ctg ggg gac aga gtc atc 720tcg gga aat tgg cat tgc gat tcc caa tgg ctg ggg gac aga gtc atc 720

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val IleSer Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240225 230 235 240

acc acc agc acc cga acc tgg gcc ctg ccc acc tac aac aat cac ctc 768acc acc agc acc cga acc tgg gcc ctg ccc acc tac aac aat cac ctc 768

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His LeuThr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255245 250 255

tac aag caa atc tcc aac agc aca tct gga gga tct tca aat gac aac 816tac aag caa atc tcc aac agc aca tct gga gga tct tca aat gac aac 816

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp AsnTyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270260 265 270

gcc tac ttc ggc tac agc acc ccc tgg ggg tat ttt gac ttc aac aga 864gcc tac ttc ggc tac agc acc ccc tgg ggg tat ttt gac ttc aac aga 864

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn ArgAla Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285275 280 285

ttc cac tgc cac ttc tca cca cgt gac tgg cag cga ctc atc aac aac 912ttc cac tgc cac ttc tca cca cgt gac tgg cag cga ctc atc aac aac 912

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn AsnPhe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300290 295 300

aac tgg gga ttc cgg cct aag cga ctc aac ttc aag ctc ttc aac att 960aac tgg gga ttc cgg cct aag cga ctc aac ttc aag ctc ttc aac att 960

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn IleAsn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320305 310 315 320

cag gtc aaa gag gtt acg gac aac aat gga gtc aag acc atc gcc aat 1008cag gtc aaa gag gtt acg gac aac aat gga gtc aag acc atc gcc aat 1008

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala AsnGln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335325 330 335

aac ctt acc agc acg gtc cag gtc ttc acg gac tca gac tat cag ctc 1056aac ctt acc agc acg gtc cag gtc ttc acg gac tca gac tat cag ctc 1056

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln LeuAsn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350340 345 350

ccg tac gtg ctc ggg tcg gct cac gag ggc tgc ctc ccg ccg ttc cca 1104ccg tac gtg ctc ggg tcg gct cac gag ggc tgc ctc ccg ccg ttc cca 1104

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe ProPro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365355 360 365

gcg gac gtt ttc atg att cct cag tac ggg tat ctg acg ctt aat gat 1152gcg gac gtt ttc atg att cct cag tac ggg tat ctg acg ctt aat gat 1152

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn AspAla Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380370 375 380

gga agc cag gcc gtg ggt cgt tcg tcc ttt tac tgc ctg gaa tat ttc 1200gga agc cag gcc gtg ggt cgt tcg tcc ttt tac tgc ctg gaa tat ttc 1200

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr PheGly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400385 390 395 400

ccg tcg caa atg cta aga acg ggt aac aac ttc cag ttc agc tac gag 1248ccg tcg caa atg cta aga acg ggt aac aac ttc cag ttc agc tac gag 1248

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr GluPro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415405 410 415

ttt gag aac gta cct ttc cat agc agc tac gct cac agc caa agc ctg 1296ttt gag aac gta cct ttc cat agc agc tac gct cac agc caa agc ctg 1296

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser LeuPhe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430420 425 430

gac cga cta atg aat cca ctc atc gac caa tac ttg tac tat ctc tca 1344gac cga cta atg aat cca ctc atc gac caa tac ttg tac tat ctc tca 1344

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu SerAsp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445435 440 445

aag act att aac ggt tct gga cag aat caa caa acg cta aaa ttc agt 1392aag act att aac ggt tct gga cag aat caa caa acg cta aaa ttc agt 1392

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe SerLys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460450 455 460

gtg gcc gga ccc agc aac atg gct gtc cag gga aga aac tac ata cct 1440gtg gcc gga ccc agc aac atg gct gtc cag gga aga aac tac ata cct 1440

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile ProVal Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480465 470 475 480

gga ccc agc tac cga caa caa cgt gtc tca acc act gtg act caa aac 1488gga ccc agc tac cga caa caa cgt gtc tca acc act gtg act caa aac 1488

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln AsnGly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495485 490 495

aac aac agc gaa ttt gct tgg cct gga gct tct tct tgg gct ctc aat 1536aac aac agc gaa ttt gct tgg cct gga gct tct tct tgg gct ctc aat 1536

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu AsnAsn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510500 505 510

gga cgt aat agc ttg atg aat cct gga cct gct atg gcc agc cac aaa 1584gga cgt aat agc ttg atg aat cct gga cct gct atg gcc agc cac aaa 1584

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His LysGly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525515 520 525

gaa gga gag gac cgt ttc ttt cct ttg tct gga tct tta att ttt ggc 1632gaa gga gag gac cgt ttc ttt cct ttg tct gga tct tta att ttt ggc 1632

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe GlyGlu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540530 535 540

aaa caa gga act gga aga gac aac gtg gat gcg gac aaa gtc atg ata 1680aaa caa gga act gga aga gac aac gtg gat gcg gac aaa gtc atg ata 1680

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met IleLys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560545 550 555 560

acc aac gaa gaa gaa att aaa act act aac ccg gta gca acg gag tcc 1728acc aac gaa gaa gaa att aaa act act aac ccg gta gca acg gag tcc 1728

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu SerThr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575565 570 575

tat gga caa gtg gcc aca aac cac cag agt gcc caa gca cag gcg cag 1776tat gga caa gtg gcc aca aac cac cag agt gcc caa gca cag gcg cag 1776

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala GlnTyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590580 585 590

acc ggc tgg gtt caa aac caa gga ata ctt ccg ggt atg gtt tgg cag 1824acc ggc tgg gtt caa aac caa gga ata ctt ccg ggt atg gtt tgg cag 1824

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp GlnThr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605595 600 605

gac aga gat gtg tac ctg caa gga ccc att tgg gcc aaa att cct cac 1872gac aga gat gtg tac ctg caa gga ccc att tgg gcc aaa att cct cac 1872

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro HisAsp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620610 615 620

acg gac ggc aac ttt cac cct tct ccg ctg atg gga ggg ttt gga atg 1920acg gac ggc aac ttt cac cct tct ccg ctg atg gga ggg ttt gga atg 1920

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly MetThr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640625 630 635 640

aag cac ccg cct cct cag atc ctc atc aaa aac aca cct gta cct gcg 1968aag cac ccg cct cct cag atc ctc atc aaa aac aca cct gta cct gcg 1968

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro AlaLys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655645 650 655

gat cct cca acg gcc ttc aac aag gac aag ctg aac tct ttc atc acc 2016gat cct cca acg gcc ttc aac aag gac aag ctg aac tct ttc atc acc 2016

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile ThrAsp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670660 665 670

cag tat tct act ggc caa gtc agc gtg gag atc gag tgg gag ctg cag 2064cag tat tct act ggc caa gtc agc gtg gag atc gag tgg gag ctg cag 2064

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu GlnGln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685675 680 685

aag gaa aac agc aag cgc tgg aac ccg gag atc cag tac act tcc aac 2112aag gaa aac agc aag cgc tgg aac ccg gag atc cag tac act tcc aac 2112

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser AsnLys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700690 695 700

tat tac aag tct aat aat gtt gaa ttt gct gtt aat act gaa ggt gta 2160tat tac aag tct aat aat gtt gaa ttt gct gtt aat act gaa ggt gta 2160

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly ValTyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720705 710 715 720

tat agt gaa ccc cgc ccc att ggc acc aga tac ctg act cgt aat ctg 2208tat agt gaa ccc cgc ccc att ggc acc aga tac ctg act cgt aat ctg 2208

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn LeuTyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735725 730 735

<210> 23<210> 23

<211> 736<211> 736

<212> PRT<212> PRT

<213> 腺相关病毒9<213> Adeno-associated virus 9

<400> 23<400> 23

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu SerMet Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 151 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln ProGlu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 3020 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu ProLys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 4535 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu ProGly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 6050 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr AspVal Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 8065 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His AlaGln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 9585 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly GlyAsp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu ProAsn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys ArgLeu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile GlyPro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln ThrLys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro ProGly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly GlyAla Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser SerAla Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val IleSer Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His LeuThr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp AsnTyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn ArgAla Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn AsnPhe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn IleAsn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala AsnGln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln LeuAsn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe ProPro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn AspAla Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr PheGly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr GluPro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser LeuPhe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu SerAsp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe SerLys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile ProVal Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln AsnGly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu AsnAsn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His LysGly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe GlyGlu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met IleLys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu SerThr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala GlnTyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp GlnThr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro HisAsp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly MetThr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro AlaLys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile ThrAsp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu GlnGln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser AsnLys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly ValTyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn LeuTyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735725 730 735

<210> 24<210> 24

<211> 130<211> 130

<212> DNA<212> DNA

<213> 腺相关病毒2<213> Adeno-associated virus 2

<400> 24<400> 24

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct 130aggggttcct 130

<210> 25<210> 25

<211> 130<211> 130

<212> DNA<212> DNA

<213> 腺相关病毒2<213> Adeno-associated virus 2

<400> 25<400> 25

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag 130gagcgcgcag 130

<210> 26<210> 26

<211> 973<211> 973

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 26<400> 26

gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg 60gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg 60

cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc 120cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccggggaggg ccctttgtgc 120

ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc 180gggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc 180

tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc 240tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc 240

agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag 300agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag 300

gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg 360gggaacaaag gctgcgtgcg gggtgtgtgc gtggggggggt gagcaggggg tgtgggcgcg 360

tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct 420tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct 420

tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg 480tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg 480

cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag 540cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcggggggag 540

gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc 600gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc 600

cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag 660cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag 660

ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc 720ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc 720

gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt 780gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt 780

ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc 840ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc 840

agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt 900agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt 900

catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat 960catgccttct tctttttcct acagctcctg ggcaacgtgc tggttatattgt gctgtctcat 960

cattttggca aag 973cattttggca aag 973

<210> 27<210> 27

<211> 589<211> 589

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 27<400> 27

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgctttccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589

<210> 28<210> 28

<211> 127<211> 127

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 28<400> 28

gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact 60gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact 60

tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 120tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 120

tcactcg 127tcactcg 127

<210> 29<210> 29

<211> 282<211> 282

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 29<400> 29

tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc 60tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccaccccc 60

caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg 120caattttgta tttatttatt ttttaattat tttgtgcagc gatggggggcg gggggggggg 120

gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag 180gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag 180

gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc 240gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc 240

ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gg 282ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gg 282

<210> 30<210> 30

<211> 382<211> 382

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成构建体<223> Synthetic constructs

<400> 30<400> 30

ctagtcgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60ctagtcgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60

atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120

cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180

tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240

tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300

ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360

acgtattagt catcgctatt ac 382acgtattagt catcgctatt ac 382

<210> 31<210> 31

<211> 22<211> 22

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> miR靶序列<223> miR target sequence

<400> 31<400> 31

agtgaattct accagtgcca ta 22agtgaattct accagtgcca ta 22

<210> 32<210> 32

<211> 24<211> 24

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<220><220>

<223> miR靶序列<223> miR target sequence

<400> 32<400> 32

agtgtgagtt ctaccattgc caaa 24agtgtgagtt ctaccattgc caaa 24

Claims (31)

Translated fromChinese

1.一种重组AAV(rAAV)，其包含其中包装有载体基因组的AAVhu68衣壳，其中所述载体基因组包含功能性人α-半乳糖苷酶A(hGLA)的编码序列和指导所述hGLA在靶细胞中表达的调节序列，1. A recombinant AAV (rAAV) comprising the AAVhu68 capsid wherein the vector genome is packaged, wherein the vector genome comprises the coding sequence of functional human α-galactosidase A (hGLA) and directs the hGLA in Regulatory sequences expressed in target cells,其中所述编码序列包含SEQ ID NO：4的核苷酸94至1287，或与其至少85％相同的序列，并且其中所述hGLA在基于SEQ ID NO：2的氨基酸残基编号的位置233和/或位置359处具有半胱氨酸残基。wherein said coding sequence comprises nucleotides 94 to 1287 of SEQ ID NO: 4, or a sequence at least 85% identical thereto, and wherein said hGLA is at position 233 based on amino acid residue numbering of SEQ ID NO: 2 and/or Or a cysteine residue at position 359.2.根据权利要求1所述的rAAV，其中所述hGLA包含SEQ ID NO：2的至少氨基酸32至429，或与其至少95％相同的序列。2. The rAAV of claim 1, wherein the hGLA comprises at least amino acids 32 to 429 of SEQ ID NO: 2, or a sequence at least 95% identical thereto.3.根据权利要求1或2所述的rAAV，其中所述hGLA包含SEQ ID NO：7的氨基酸32至429。3. The rAAV of claim 1 or 2, wherein the hGLA comprises amino acids 32 to 429 of SEQ ID NO:7.4.根据权利要求1至3中任一项所述的rAAV，其中所述hGLA包含天然信号肽。4. The rAAV of any one of claims 1 to 3, wherein the hGLA comprises a native signal peptide.5.根据权利要求1至3中任一项所述的rAAV，其中所述hGLA包含异源信号肽。5. The rAAV of any one of claims 1 to 3, wherein the hGLA comprises a heterologous signal peptide.6.根据权利要求1至4中任一项所述的rAAV，其中所述hGLA包含SEQ ID NO：17的全长(氨基酸1至429)，或与其至少95％相同的序列。6. The rAAV according to any one of claims 1 to 4, wherein said hGLA comprises the full length of SEQ ID NO: 17 (amino acids 1 to 429), or a sequence at least 95% identical thereto.7.根据权利要求1至6中任一项所述的rAAV，其中所述载体基因组包含组织特异性启动子。7. The rAAV of any one of claims 1 to 6, wherein the vector genome comprises a tissue-specific promoter.8.根据权利要求1至6中任一项所述的rAAV，其中所述调节序列包含CB7启动子、内含子和polyA。8. The rAAV of any one of claims 1 to 6, wherein the regulatory sequence comprises a CB7 promoter, an intron, and polyA.9.根据权利要求1至8中任一项所述的rAAV，其中所述调节序列包含土拨鼠肝炎病毒转录后调节元件(WPRE)。9. The rAAV of any one of claims 1 to 8, wherein the regulatory sequence comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).10.根据权利要求9所述的rAAV，其中所述WPRE包含SEQ ID NO：27。10. The rAAV of claim 9, wherein the WPRE comprises SEQ ID NO:27.11.根据权利要求1至9中任一项所述的rAAV，其中所述载体基因组包含一个或多个miRNA靶序列。11. The rAAV of any one of claims 1 to 9, wherein the vector genome comprises one or more miRNA target sequences.12.根据权利要求1所述的rAAV，其中所述载体基因组包含与SEQ ID NO：6至少85％相同的序列。12. The rAAV of claim 1, wherein the vector genome comprises a sequence at least 85% identical to SEQ ID NO:6.13.一种表达盒，其包含编码功能性人α-半乳糖苷酶A(hGLA)的核酸序列和指导所述hGLA在含有所述表达盒的靶细胞中表达的一个或多个调节序列，13. An expression cassette comprising a nucleic acid sequence encoding functional human alpha-galactosidase A (hGLA) and one or more regulatory sequences directing the expression of said hGLA in target cells containing said expression cassette,其中所述核酸序列包含SEQ ID NO：4的核苷酸94至1287，或与其至少85％相同的序列，并且其中所述hGLA在基于SEQ ID NO：2或SEQ ID NO：7的氨基酸残基编号的位置233和/或位置359处具有半胱氨酸残基。wherein said nucleic acid sequence comprises nucleotides 94 to 1287 of SEQ ID NO: 4, or a sequence at least 85% identical thereto, and wherein said hGLA is based on amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 7 Numbered positions 233 and/or 359 have cysteine residues.14.根据权利要求12所述的表达盒，其中所述hGLA包含SEQ ID NO：7的氨基酸32至429。14. The expression cassette of claim 12, wherein the hGLA comprises amino acids 32 to 429 of SEQ ID NO:7.15.根据权利要求12或13所述的表达盒，其中所述hGLA包含天然信号肽。15. The expression cassette according to claim 12 or 13, wherein said hGLA comprises a native signal peptide.16.根据权利要求12至14中任一项所述的表达盒，其中所述hGLA包含异源信号肽。16. The expression cassette according to any one of claims 12 to 14, wherein said hGLA comprises a heterologous signal peptide.17.根据权利要求12至15中任一项所述的表达盒，其中所述hGLA包含SEQ ID NO：7的全长(氨基酸1至429)，或与其至少95％相同的序列。17. The expression cassette according to any one of claims 12 to 15, wherein said hGLA comprises the full length of SEQ ID NO: 7 (amino acids 1 to 429), or a sequence at least 95% identical thereto.18.根据权利要求12至16中任一项所述的表达盒，其中所述表达盒包含组织特异性启动子。18. The expression cassette according to any one of claims 12 to 16, wherein the expression cassette comprises a tissue specific promoter.19.根据权利要求12至16中任一项所述的表达盒，其中所述调节序列包含CB7启动子、内含子和polyA。19. The expression cassette according to any one of claims 12 to 16, wherein the regulatory sequence comprises a CB7 promoter, an intron and polyA.20.根据权利要求12至18中任一项所述的表达盒，其中所述调节序列包含土拨鼠肝炎病毒转录后调节元件(WPRE)。20. The expression cassette according to any one of claims 12 to 18, wherein the regulatory sequence comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).21.根据权利要求19所述的rAAV，其中所述WPRE包含SEQ ID NO：27。21. The rAAV of claim 19, wherein the WPRE comprises SEQ ID NO:27.22.根据权利要求12至20中任一项所述的表达盒，其进一步包含一个或多个miRNA靶序列。22. The expression cassette according to any one of claims 12 to 20, further comprising one or more miRNA target sequences.23.根据权利要求12至21中任一项所述的表达盒，其中所述表达盒由非病毒载体或病毒载体携带。23. The expression cassette according to any one of claims 12 to 21, wherein the expression cassette is carried by a non-viral vector or a viral vector.24.根据权利要求22所述的表达盒，其中非病毒载体选自裸DNA、裸RNA、质粒、无机颗粒、脂质颗粒、基于聚合物的载体或基于壳聚糖的制剂。24. The expression cassette according to claim 22, wherein the non-viral vector is selected from naked DNA, naked RNA, plasmids, inorganic particles, lipid particles, polymer-based carriers or chitosan-based formulations.25.根据权利要求24所述的表达盒，其中病毒载体是重组细小病毒、重组慢病毒、重组逆转录病毒、重组腺病毒。25. The expression cassette according to claim 24, wherein the viral vector is a recombinant parvovirus, a recombinant lentivirus, a recombinant retrovirus, a recombinant adenovirus.26.一种包含根据权利要求12至24中任一项所述的表达盒的质粒，任选地其中所述表达盒的侧翼为AAV 5′ITR和AAV 3′ITR。26. A plasmid comprising an expression cassette according to any one of claims 12 to 24, optionally wherein the expression cassette is flanked by AAV 5'ITR and AAV 3'ITR.27.一种宿主细胞，其包含根据权利要求12至24中任一项所述的表达盒或根据权利要求25所述的质粒。27. A host cell comprising the expression cassette according to any one of claims 12 to 24 or the plasmid according to claim 25.28.一种药物组合物，其包含根据权利要求1至11中任一项所述的rAAV或根据权利要求12至24中任一项所述的表达盒，和药学上可接受的载体。28. A pharmaceutical composition comprising the rAAV according to any one of claims 1 to 11 or the expression cassette according to any one of claims 12 to 24, and a pharmaceutically acceptable carrier.29.一种治疗诊断为GLA缺乏症(法布里病)的人类受试者的方法，所述方法包含向所述受试者施用根据权利要求1至11中任一项所述的rAAV、根据权利要求12至24中任一项所述的表达盒或根据权利要求27所述的药物组合物。29. A method of treating a human subject diagnosed with GLA deficiency (Fabry disease), said method comprising administering to said subject an rAAV according to any one of claims 1 to 11, An expression cassette according to any one of claims 12 to 24 or a pharmaceutical composition according to claim 27.30.根据权利要求1至11中任一项所述的rAAV、根据权利要求12至24中任一项所述的表达盒或根据权利要求27所述的药物组合物，其用于治疗GLA缺乏症(法布里病)。30. The rAAV according to any one of claims 1 to 11, the expression cassette according to any one of claims 12 to 24 or the pharmaceutical composition according to claim 27 for use in the treatment of GLA deficiency syndrome (Fabry disease).31.根据权利要求1至11中任一项所述的rAAV、根据权利要求12至24中任一项所述的表达盒或根据权利要求27所述的药物组合物，其用于制造用于治疗GLA缺乏症(法布里病)的药物。31. The rAAV according to any one of claims 1 to 11, the expression cassette according to any one of claims 12 to 24, or the pharmaceutical composition according to claim 27 for the manufacture of Medicines to treat GLA deficiency (Fabry disease).