Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway

Matthew L. Hirsch, Chengwen Li, Isabella Bellon, Chaoying Yin, Sai Chavala, Marina Pryadkina, Isabelle Richard, Richard Jude Samulski

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

A drawback of gene therapy using adeno-associated virus (AAV) is the DNA packaging restriction of the viral capsid (<4.7 kb). Recent observations demonstrate oversized AAV genome transduction through an unknown mechanism. Herein, AAV production using an oversized reporter (6.2 kb) resulted in chloroform and DNase-resistant particles harboring distinct "fragment" AAV (fAAV) genomes (5.0, 2.4, and 1.6 kb). Fractionation experiments determined that only the larger "fragments" mediated transduction in vitro, and relatively efficient transduction was also demonstrated in the muscle, the eye, and the liver. In contrast with concatemerization-dependent large-gene delivery by split AAV, fAAV transduction is independent of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo while disproportionately reliant on the DNA strand-annealing protein Rad51C. Importantly, fAAV's unique dependence on DNA repair proteins, compared with intact AAV, strongly suggests that the majority of oversized AAV transduction is mediated by fragmented genomes. Although fAAV transduction is less efficient than intact AAV, it is enhanced fourfold in muscle and sevenfold in the retina compared with split AAV transduction. Furthermore, fAAV carrying codon-optimized therapeutic dysferlin cDNA in a 7.5 kb expression cassette restored dysferlin levels in a dystrophic model. Collectively, oversized AAV genome transduction requires unique DNA repair pathways and offers an alternative, more efficient strategy for large-gene therapy.

Original languageEnglish
Pages (from-to)2205-2216
Number of pages12
JournalMolecular Therapy
Volume21
Issue number12
DOIs
StatePublished - 1 Jan 2013

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Dependovirus
DNA
Genome
DNA Repair
Genetic Therapy
DNA-Activated Protein Kinase
DNA Packaging
Catalytic DNA
Muscles
Deoxyribonucleases
Capsid
Chloroform
Codon
Retina
Catalytic Domain
Proteins
Complementary DNA

Cite this

Hirsch, M. L., Li, C., Bellon, I., Yin, C., Chavala, S., Pryadkina, M., ... Samulski, R. J. (2013). Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway. Molecular Therapy, 21(12), 2205-2216. https://doi.org/10.1038/mt.2013.184
Hirsch, Matthew L. ; Li, Chengwen ; Bellon, Isabella ; Yin, Chaoying ; Chavala, Sai ; Pryadkina, Marina ; Richard, Isabelle ; Samulski, Richard Jude. / Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway. In: Molecular Therapy. 2013 ; Vol. 21, No. 12. pp. 2205-2216.
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Hirsch, ML, Li, C, Bellon, I, Yin, C, Chavala, S, Pryadkina, M, Richard, I & Samulski, RJ 2013, 'Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway', Molecular Therapy, vol. 21, no. 12, pp. 2205-2216. https://doi.org/10.1038/mt.2013.184

Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway. / Hirsch, Matthew L.; Li, Chengwen; Bellon, Isabella; Yin, Chaoying; Chavala, Sai; Pryadkina, Marina; Richard, Isabelle; Samulski, Richard Jude.

In: Molecular Therapy, Vol. 21, No. 12, 01.01.2013, p. 2205-2216.

Research output: Contribution to journalArticle

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T1 - Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway

AU - Hirsch, Matthew L.

AU - Li, Chengwen

AU - Bellon, Isabella

AU - Yin, Chaoying

AU - Chavala, Sai

AU - Pryadkina, Marina

AU - Richard, Isabelle

AU - Samulski, Richard Jude

PY - 2013/1/1

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AB - A drawback of gene therapy using adeno-associated virus (AAV) is the DNA packaging restriction of the viral capsid (<4.7 kb). Recent observations demonstrate oversized AAV genome transduction through an unknown mechanism. Herein, AAV production using an oversized reporter (6.2 kb) resulted in chloroform and DNase-resistant particles harboring distinct "fragment" AAV (fAAV) genomes (5.0, 2.4, and 1.6 kb). Fractionation experiments determined that only the larger "fragments" mediated transduction in vitro, and relatively efficient transduction was also demonstrated in the muscle, the eye, and the liver. In contrast with concatemerization-dependent large-gene delivery by split AAV, fAAV transduction is independent of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo while disproportionately reliant on the DNA strand-annealing protein Rad51C. Importantly, fAAV's unique dependence on DNA repair proteins, compared with intact AAV, strongly suggests that the majority of oversized AAV transduction is mediated by fragmented genomes. Although fAAV transduction is less efficient than intact AAV, it is enhanced fourfold in muscle and sevenfold in the retina compared with split AAV transduction. Furthermore, fAAV carrying codon-optimized therapeutic dysferlin cDNA in a 7.5 kb expression cassette restored dysferlin levels in a dystrophic model. Collectively, oversized AAV genome transduction requires unique DNA repair pathways and offers an alternative, more efficient strategy for large-gene therapy.

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