EFFICIENCY AND SAFETY OF AAV MEDIATED GENE DELIVERY OF THE HUMAN ND4 COMPLEX I SUBUNIT IN THE MOUSE VISUAL SYSTEM. Export

@article{citeulike:4732070, abstract = {PURPOSE: To evaluate the efficiency and safety of AAV mediated gene delivery of a normal human ND4 complex I subunit in the mouse visual system. METHODS: A nuclear encoded human ND4 subunit fused to the ATPc mitochondrial targeting sequence and FLAG epitope were packaged in AAV2 capsids that were injected into the right eyes of mice. AAV-GFP was injected into the left eyes. A month later, PERGs, rate of ATP synthesis, gene expression and incorporation of the human ND4 subunit into the murine complex I were evaluated. Quantitative analysis of ND4FLAG injected eyes was assessed relative to GFP injected eyes. RESULTS: The rate of ATP synthesis and PERG amplitudes were similar in ND4FLAG and GFP inoculated eyes. PERG latency was shorter in eyes that received ND4FLAG. Immunoprecipitated murine complex I gave the expected 52 kDa band of processed human ND4FLAG. Confocal microscopy revealed perinuclear expression of FLAG co-localized with MitoTracker. Transmission electron microscopy revealed FLAG immungold within mitochondria. Relative to Thy1.2-positive RGCs, quantification of FLAG-positive RGCs was 38\% and GFP-positive RGCs was 65\%. Thy1.2 positive-RGC counts in AAV-ND4FLAG were similar to control eyes injected with AAV-GFP. CONCLUSIONS: Human ND4 was properly processed and imported into mitochondria of ganglion cells of the retina and axons of the optic nerve of mice following intravitreal injection. Although approximately two-thirds the efficiency of GFP, expression of normal human ND4 in murine mitochondria did not induce loss of RGCs, ATP synthesis or PERG amplitude, suggesting that allotopic ND4 may be safe for treatment of LHON.}, author = {Guy, John and Qi, Xiaoping and Koilkonda, Rajeshwari D D. and Arguello, Tania and Chou, Tsung-Han H. and Ruggeri, Marco and Porciatti, Vittorio and Lewin, Alfred S S. and Hauswirth, William W W.}, citeulike-article-id = {4732070}, citeulike-linkout-0 = {http://dx.doi.org/10.1167/iovs.08-3214}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/19387075}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=19387075}, day = {22}, doi = {10.1167/iovs.08-3214}, issn = {1552-5783}, journal = {Investigative ophthalmology \& visual science}, keywords = {allotopic, example, human, mitochondria, mouse, projmt}, month = {April}, posted-at = {2009-06-03 12:54:21}, priority = {4}, title = {EFFICIENCY AND SAFETY OF AAV MEDIATED GENE DELIVERY OF THE HUMAN ND4 COMPLEX I SUBUNIT IN THE MOUSE VISUAL SYSTEM.}, url = {http://dx.doi.org/10.1167/iovs.08-3214}, year = {2009} }

TY - JOUR ID - citeulike:4732070 L3 - citeulike-article-id:4732070 N2 - PURPOSE: To evaluate the efficiency and safety of AAV mediated gene delivery of a normal human ND4 complex I subunit in the mouse visual system. METHODS: A nuclear encoded human ND4 subunit fused to the ATPc mitochondrial targeting sequence and FLAG epitope were packaged in AAV2 capsids that were injected into the right eyes of mice. AAV-GFP was injected into the left eyes. A month later, PERGs, rate of ATP synthesis, gene expression and incorporation of the human ND4 subunit into the murine complex I were evaluated. Quantitative analysis of ND4FLAG injected eyes was assessed relative to GFP injected eyes. RESULTS: The rate of ATP synthesis and PERG amplitudes were similar in ND4FLAG and GFP inoculated eyes. PERG latency was shorter in eyes that received ND4FLAG. Immunoprecipitated murine complex I gave the expected 52 kDa band of processed human ND4FLAG. Confocal microscopy revealed perinuclear expression of FLAG co-localized with MitoTracker. Transmission electron microscopy revealed FLAG immungold within mitochondria. Relative to Thy1.2-positive RGCs, quantification of FLAG-positive RGCs was 38% and GFP-positive RGCs was 65%. Thy1.2 positive-RGC counts in AAV-ND4FLAG were similar to control eyes injected with AAV-GFP. CONCLUSIONS: Human ND4 was properly processed and imported into mitochondria of ganglion cells of the retina and axons of the optic nerve of mice following intravitreal injection. Although approximately two-thirds the efficiency of GFP, expression of normal human ND4 in murine mitochondria did not induce loss of RGCs, ATP synthesis or PERG amplitude, suggesting that allotopic ND4 may be safe for treatment of LHON. JF - Investigative ophthalmology & visual science SN - 1552-5783 TI - EFFICIENCY AND SAFETY OF AAV MEDIATED GENE DELIVERY OF THE HUMAN ND4 COMPLEX I SUBUNIT IN THE MOUSE VISUAL SYSTEM. KW - allotopic KW - example KW - human KW - mitochondria KW - mouse KW - projmt AU - Guy, John AU - Qi, Xiaoping AU - Koilkonda, Rajeshwari D AU - Arguello, Tania AU - Chou, Tsung-Han AU - Ruggeri, Marco AU - Porciatti, Vittorio AU - Lewin, Alfred S AU - Hauswirth, William W PY - 2009/04/22/ UR - http://dx.doi.org/10.1167/iovs.08-3214 ER -