title: Construction and detection of a novel type of recombinant human rAAV2/2-ND4.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.
authors.affiliation: Department of Ophthalmology, Tongji Medical College, Huazhong University of Science & Technology, 430030 Wuhan, China.
authors.forename: Han
authors.initials: H
authors.lastname: Pei
authors.affiliation: Department of Ophthalmology, Tongji Medical College, Huazhong University of Science & Technology, 430030 Wuhan, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Wan
authors.affiliation: Department of Ophthalmology, Tongji Medical College, Huazhong University of Science & Technology, 430030 Wuhan, China.
authors.forename: Weikun
authors.initials: W
authors.lastname: Hu
authors.affiliation: Department of Ophthalmology, Tongji Medical College, Huazhong University of Science & Technology, 430030 Wuhan, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Dong
authors.affiliation: Department of Ophthalmology, Tongji Medical College, Huazhong University of Science & Technology, 430030 Wuhan, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
mesh_terms: D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use; D006801:Humans; D008928:Mitochondria / Q000235:genetics; D009245:NADH Dehydrogenase / Q000235:genetics* / Q000627:therapeutic use; D029242:Optic Atrophy, Hereditary, Leber / Q000235:genetics / Q000628:therapy*
chemical_list: C098483:NADH dehydrogenase subunit 4; D009245:NADH Dehydrogenase