Chen Min, Maeng Kyungah, Nawab Akbar, Francois Rony A, Bray Julie K, Reinhard Mary K, Boye Sanford L, Hauswirth William W, Kaye Frederic J, Aslanidi Georgiy, Srivastava Arun, Zajac-Kaye Maria
1 Department of Anatomy and Cell Biology, University of Florida College of Medicine , Gainesville, Florida.
2 Department of Veterinary Medicine, University of Florida College of Medicine , Gainesville, Florida.
Hum Gene Ther Methods. 2017 Feb;28(1):49-59. doi: 10.1089/hgtb.2016.089.
Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to Kras, Kras/Pten, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45-70%) compared with WT-AAV8 (7%; p < 0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared with WT-AAV8 (10,285 vs. 1,500 vector copies/μg DNA respectively, p < 0.05). In addition, we observed that intraperitoneal injection of the double-mutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding ∼14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/μg DNA respectively, p < 0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular.
尽管人们努力使用腺相关病毒（AAV）载体介导的基因疗法来治疗胰腺导管腺癌（PDAC），但转导效率仍然是一个限制因素，因此提高AAV递送效率将显著促进这种恶性肿瘤的治疗。据报道，将各种AAV血清型衣壳表面特定的酪氨酸（Y）残基定点突变为苯丙氨酸（F）是提高基因转移效率的一种方法。在本研究中，我们确定Y到F的突变是否也能增强胰腺中AAV8的基因转移，以促进PDAC的基因治疗。通过腹腔内或尾静脉途径，将三种不同的Y到F突变载体（一种单突变体Y733F；一种双突变体Y447F+Y733F；以及一种三突变体Y275F+Y447F+Y733F）和野生型AAV8（WT-AAV8）分别注射到Kras、Kras/Pten和野生型小鼠体内。给药2周后，在胰腺或PDAC中评估这些表达mCherry报告基因的载体的转导效率，并将其与病毒基因组拷贝数相关联。我们对转导谱的比较和定量分析表明，与WT-AAV8（7%；p<0.01）相比，Y到F双突变体在胰腺组织中表现出最高的mCherry表达（范围为45-70%）。与WT-AAV8相比，用双突变体AAV8转导后，我们在正常胰腺中还检测到载体基因组拷贝数水平高出7倍（分别为10285和1500个载体拷贝/μg DNA，p<0.05）。此外，我们观察到，与WT-AAV8相比，腹腔注射双突变体AAV8在小鼠PDAC中的转导效率提高了15倍，载体基因组拷贝数相应增加了约14倍（分别为26575和2165个拷贝/μg DNA，p<0.05）。这些发现表明，Y447+Y733F-AAV8能显著提高正常和恶性胰腺组织中的转导效率，这表明该载体在一般靶向胰腺疾病，特别是PDAC方面具有潜在应用价值。
