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Updated: May 4, 2026
Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors
Published on: April 5, 2024
Construction and detection of a novel type of recombinant human rAAV2/2-ND4
Han Pei1, Xing Wan1, Weikun Hu1
1Department of Ophthalmology, Tongji Medical College, Huazhong University of Science & Technology, 430030 Wuhan, China.
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.
