Sen Dwaipayan, Gadkari Rupali A, Sudha Govindarajan, Gabriel Nishanth, Kumar Yesupatham Sathish, Selot Ruchita, Samuel Rekha, Rajalingam Sumathi, Ramya V, Nair Sukesh C, Srinivasan Narayanaswamy, Srivastava Alok, Jayandharan Giridhara R
Department of Hematology, Christian Medical College, Vellore 632004, Tamil Nadu, India.
Hum Gene Ther Methods. 2013 Apr;24(2):104-16. doi: 10.1089/hgtb.2012.195.
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 (~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 biodistribution 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.
基于8型血清型（AAV8）的重组腺相关病毒载体在肝脏定向基因治疗方面显示出巨大潜力。然而，为了克服AAV8载体所见的载体剂量依赖性免疫毒性，开发在显著低载体剂量下能提供增强基因表达的更好的AAV8载体很重要。由于已知AAV载体在细胞内运输过程中会被宿主细胞激酶/泛素化/蛋白酶体机制靶向在细胞质中破坏，我们对AAV8衣壳上的特定丝氨酸/苏氨酸激酶或泛素化靶点进行了修饰，以提高其转导效率。在AAV8衣壳中与先前成功产生的有效AAV2突变体等效的位置引入特定丝氨酸（S）/苏氨酸（T）/赖氨酸（K）残基的点突变。随后进行了广泛的结构分析，以评估两种血清型之间的结构等效性。对具有野生型（WT）以及每个S/T→丙氨酸（A）或K→精氨酸（R）突变体衣壳的单链AAV8载体在C57BL/6小鼠体内的肝脏转导效率进行了评估。与仅接受WT-AAV8载体的动物相比，两个AAV8-S→A突变体（S279A和S671A）以及一个K137R突变体载体在肝脏中显示出显著更高的增强型绿色荧光蛋白（EGFP）转录水平（约9至46倍）。表现最佳的AAV8突变体（K137R）载体的病毒衣壳泛素化也显著降低，先天免疫反应标志物的激活减少，并且与WT-AAV8载体相比，中和抗体形成水平相应降低了两倍。载体生物分布研究表明，与WT-AAV8载体相比，K137R突变体在肝脏中的转导显著更高且更具特异性（106对7.7载体拷贝/小鼠二倍体基因组）。为了进一步研究K137R-AAV8突变体在治疗性基因转移中的效用，我们在肝脏特异性启动子（LP1或hAAT）的控制下将人凝血因子IX（h.FIX）递送至C57BL/6小鼠体内。在肝脏基因转移后长达8周时，所有K137R-AAV8处理组中的h.FIX:Ag循环水平均较高。这些研究证明了使用这种新型AAV8载体进行B型血友病潜在基因治疗的可行性。
