Yu Hong, Mehta Arpit, Wang Gaofeng, Hauswirth William W, Chiodo Vince, Boye Sanford L, Guy John
Bascom Palmer Eye Institute, University of Miami, Miami, FL 33136, USA.
Mol Vis. 2013 Jul 14;19:1482-91. Print 2013.
To determine the effects of mitochondrial targeting sequence (MTS) modified AAV gene delivery of wild-type human NADH dehydrogenase subunit 4 (ND4), mutated in most cases of the blinding disease Leber hereditary optic neuropathy (LHON), on the host mouse mitochondrial genome.
We injected a modified self-complementary (sc) AAV vector, to which we appended the cytochrome oxidase subunit 8 (COX8) leader to one of the three capsid proteins (VP2) comprising the protein shell of the AAV virion, into the mouse vitreous to deliver the human ND4 gene under the control of a mitochondrial heavy strand promoter (HSP) directly to the mitochondria of the mouse retina. Control viruses consisting of scAAV lacking the COX8 targeting sequence and containing human ND4, or scAAV containing GFP, were also vitreally injected. Using next-generation sequencing of mitochondrial DNA extracted from the pooled mouse retinas of experimental and control eyes, we tested for the presence of the transferred human ND4, and any potential recombination of the transferred human ND4 gene with the endogenous host mitochondrial genome.
We found hundreds of human ND4 DNA reads in mitochondrial samples of MTS AAV-ND4-injected eyes, a few human ND4 reads with AAV-ND4 lacking the MTS, and none with AAV-GFP injection. Putative chimeric read pairs at the 5' or 3' ends of human ND4 showed only vector sequences without the flanking mouse sequences expected with homologous recombination of human ND4 with the murine ND4. Examination of mouse mitochondrial ND4 sequences for evidence of intra-molecular small-scale homologous recombination events yielded no significant stretches greater than three to four nucleotides attributable to human ND4. Furthermore, in no instance did human ND4 insert into other non-homologous sites of the 16 kb host mtDNA.
Our findings suggest that human ND4 remains episomal in host mitochondria and is not disruptive to any of the endogenous mitochondrial genes of the host genome. Therefore, mitochondrial gene transfer with an MTS-AAV is non-mutagenic and likely to be safe if used to treat LHON patients with mutated ND4.
确定线粒体靶向序列（MTS）修饰的腺相关病毒（AAV）介导野生型人类烟酰胺腺嘌呤二核苷酸脱氢酶亚基4（ND4）基因传递对宿主小鼠线粒体基因组的影响。ND4在大多数致盲性疾病——Leber遗传性视神经病变（LHON）中发生突变。
我们将一种修饰的自互补（sc）AAV载体注入小鼠玻璃体，该载体在构成AAV病毒粒子蛋白外壳的三种衣壳蛋白之一（VP2）上附加了细胞色素氧化酶亚基8（COX8）前导序列，以在线粒体重链启动子（HSP）的控制下将人类ND4基因直接递送至小鼠视网膜的线粒体。还玻璃体注射了对照病毒，包括缺乏COX8靶向序列且含有人类ND4的scAAV，或含有绿色荧光蛋白（GFP）的scAAV。通过对从实验和对照眼的合并小鼠视网膜中提取的线粒体DNA进行下一代测序，我们检测了转移的人类ND4的存在，以及转移的人类ND4基因与内源性宿主线粒体基因组的任何潜在重组。
我们在注射MTS AAV-ND4的眼睛的线粒体样本中发现了数百条人类ND4 DNA读数，在缺乏MTS的AAV-ND4样本中有少量人类ND4读数，而在注射AAV-GFP的样本中未发现。在人类ND4的5'或3'端假定的嵌合读数对仅显示载体序列，没有预期的人类ND4与小鼠ND4同源重组时的侧翼小鼠序列。检查小鼠线粒体ND4序列中分子内小规模同源重组事件的证据，未发现大于三到四个核苷酸的明显延伸可归因于人类ND4。此外，人类ND4从未插入16 kb宿主线粒体DNA的其他非同源位点。
我们的研究结果表明，人类ND4在宿主线粒体中保持游离状态，不会破坏宿主基因组的任何内源性线粒体基因。因此，使用MTS-AAV进行线粒体基因转移不会产生诱变作用，如果用于治疗携带突变ND4的LHON患者可能是安全的。
