Next-generation sequencing of mitochondrial targeted AAV transfer of human ND4 in mice

Hong Yu, Arpit Mehta, Gaofeng Wang, William W. Hauswirth, Vince Chiodo, Sanford L. Boye, John Guy

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.

Original languageEnglish
Pages (from-to)1482-1491
Number of pages10
JournalMolecular Vision
Volume19
StatePublished - Jul 14 2013

Fingerprint

NADH dehydrogenase subunit 4
Leber's Hereditary Optic Atrophy
Mitochondrial Genome
Mitochondrial Genes
Homologous Recombination
Electron Transport Complex IV
Mitochondrial DNA
Retina
Mitochondria
Genes
Capsid Proteins
Virion
Genetic Recombination
Nucleotides
Genome
Viruses
Injections
DNA
Proteins

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Yu, H., Mehta, A., Wang, G., Hauswirth, W. W., Chiodo, V., Boye, S. L., & Guy, J. (2013). Next-generation sequencing of mitochondrial targeted AAV transfer of human ND4 in mice. Molecular Vision, 19, 1482-1491.

Next-generation sequencing of mitochondrial targeted AAV transfer of human ND4 in mice. / Yu, Hong; Mehta, Arpit; Wang, Gaofeng; Hauswirth, William W.; Chiodo, Vince; Boye, Sanford L.; Guy, John.

In: Molecular Vision, Vol. 19, 14.07.2013, p. 1482-1491.

Research output: Contribution to journalArticle

Yu, H, Mehta, A, Wang, G, Hauswirth, WW, Chiodo, V, Boye, SL & Guy, J 2013, 'Next-generation sequencing of mitochondrial targeted AAV transfer of human ND4 in mice', Molecular Vision, vol. 19, pp. 1482-1491.
Yu H, Mehta A, Wang G, Hauswirth WW, Chiodo V, Boye SL et al. Next-generation sequencing of mitochondrial targeted AAV transfer of human ND4 in mice. Molecular Vision. 2013 Jul 14;19:1482-1491.
Yu, Hong ; Mehta, Arpit ; Wang, Gaofeng ; Hauswirth, William W. ; Chiodo, Vince ; Boye, Sanford L. ; Guy, John. / Next-generation sequencing of mitochondrial targeted AAV transfer of human ND4 in mice. In: Molecular Vision. 2013 ; Vol. 19. pp. 1482-1491.
@article{fcdeec761d314e8baaf5c0166e83af90,
title = "Next-generation sequencing of mitochondrial targeted AAV transfer of human ND4 in mice",
abstract = "Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.",
author = "Hong Yu and Arpit Mehta and Gaofeng Wang and Hauswirth, {William W.} and Vince Chiodo and Boye, {Sanford L.} and John Guy",
year = "2013",
month = "7",
day = "14",
language = "English",
volume = "19",
pages = "1482--1491",
journal = "Molecular Vision",
issn = "1090-0535",

}

TY - JOUR

T1 - Next-generation sequencing of mitochondrial targeted AAV transfer of human ND4 in mice

AU - Yu, Hong

AU - Mehta, Arpit

AU - Wang, Gaofeng

AU - Hauswirth, William W.

AU - Chiodo, Vince

AU - Boye, Sanford L.

AU - Guy, John

PY - 2013/7/14

Y1 - 2013/7/14

N2 - Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.

AB - Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.

UR - http://www.scopus.com/inward/record.url?scp=84880310566&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84880310566&partnerID=8YFLogxK

M3 - Article

C2 - 23869167

AN - SCOPUS:84880310566

VL - 19

SP - 1482

EP - 1491

JO - Molecular Vision

JF - Molecular Vision

SN - 1090-0535

ER -