Han Pei1, Xing Wan1, Weikun Hu1, Xiaoyan Dong1, Bin Li1. 1. Department of Ophthalmology, Tongji Medical College, Huazhong University of Science & Technology, 430030 Wuhan, China.
Abstract
PURPOSE: To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic neuropathy (LHON). METHODS: A novel type of normal human ND4 gene was synthesized artificially to contain a mitochondrial targeting sequence that induces the translocation of this gene into mitochondria. This recombinant adeno-associated virus type 2/ serotype 2 (rAAV2/2)-mediated NADH dehydrogenase subunit 4 (ND4) gene was constructed, purified, condensed, and amplified by PCR. The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigenin-labeled H1 probe. Expression of ND4 in mitochondria was evaluated by immunofluorescence. RESULTS: The constructed rAAV2/2-ND4 specifically amplified the target gene band of ND4 and the physical titer of ND4 gene was 1.0x 10(11) vg/mL, confirming that the recombinant adenovirus vector contained the ND4 target gene. Expression of the ND4 gene was detected in mitochondria by immunofluorescence. CONCLUSION: A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.
PURPOSE: To construct a novel AAV-mediated gene delivery of the humanND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic neuropathy (LHON). METHODS: A novel type of normal humanND4 gene was synthesized artificially to contain a mitochondrial targeting sequence that induces the translocation of this gene into mitochondria. This recombinant adeno-associated virus type 2/ serotype 2 (rAAV2/2)-mediated NADH dehydrogenase subunit 4 (ND4) gene was constructed, purified, condensed, and amplified by PCR. The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigenin-labeled H1 probe. Expression of ND4 in mitochondria was evaluated by immunofluorescence. RESULTS: The constructed rAAV2/2-ND4 specifically amplified the target gene band of ND4 and the physical titer of ND4 gene was 1.0x 10(11) vg/mL, confirming that the recombinant adenovirus vector contained the ND4 target gene. Expression of the ND4 gene was detected in mitochondria by immunofluorescence. CONCLUSION: A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.