Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo
Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo
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DOI：
10.1089/hgtb.2012.195
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发表时间：
2013-04-01
影响因子：
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通讯作者：
Jayandharan, Giridhara R.
中科院分区：
文献类型：
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作者：
Sen, Dwaipayan;Gadkari, Rupali A.;Jayandharan, Giridhara R.
基于血清型8（AAV 8）的重组腺相关病毒载体已经显示出用于肝脏定向基因治疗的显著前景。然而，为了克服用AAV 8载体观察到的载体剂量依赖性免疫毒性，重要的是开发在显著低的载体剂量下提供增强的基因表达的更好的AAV 8载体。由于已知AAV载体在细胞内运输期间被宿主细胞激酶/泛素化/蛋白酶体机制靶向在细胞质中破坏，我们修饰了AAV 8衣壳上的特定丝氨酸/苏氨酸激酶或泛素化靶标以增强其转导效率。将特定丝氨酸（S）/苏氨酸（T）/赖氨酸（K）残基处的点突变引入AAV 8衣壳中与先前成功产生的有效AAV 2突变体的位置等同的位置。随后进行了广泛的结构分析，以评价两种血清型之间的结构等效性。在C57 BL/6小鼠体内评价具有野生型（WT）和S/T ->丙氨酸（A）或K-精氨酸（R）突变体衣壳中的每一种的scAAV 8载体的肝转导效率。与仅接受WT-AAV 8载体的动物相比，两种AAV 8-S -> A突变体（S279 A和S671 A）和K137 R突变体载体在肝脏中显示出显著更高的增强的绿色荧光蛋白（EGFP）转录物水平（类似于9至46倍）。与WT-AAV 8载体相比，表现最佳的AAV 8突变体（K137 R）载体还具有显著降低的病毒衣壳的泛素化，降低的先天免疫应答标志物的活化，以及伴随的中和抗体形成水平的两倍降低。载体生物分布研究显示，当与WT-AAV 8载体相比时，K137 R突变体具有显著更高和优先的肝脏转导（106对7.7载体拷贝/小鼠二倍体基因组）。为了进一步研究K137 R-AAV 8突变体在治疗性基因转移中的效用，我们将人凝血因子IX（h. FIX）在肝特异性启动子（LP 1或hAAT）的控制下导入C57 BL/6小鼠。血中h. FIX：Ag在所有K137 R-AAV 8处理组中均较高，直至肝基因转移后8周。这些研究证明了使用这种新型AAV 8载体用于血友病B的潜在基因治疗的可行性。
Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T -> Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S -> A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (similar to 9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h. FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h. FIX: Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.