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

Mol Ther. 2013 Dec;21(12):2205-16. doi: 10.1038/mt.2013.184. Epub 2013 Aug 13.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Capsid / metabolism*
  • Cricetulus
  • DNA Packaging
  • DNA, Viral / genetics
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Dependovirus / genetics
  • Dependovirus / physiology*
  • Disease Models, Animal
  • Eye / virology
  • Genes, Reporter
  • Genetic Therapy*
  • Genetic Vectors
  • Genome, Viral
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Liver / virology*
  • Mice
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism
  • Muscle, Skeletal / virology
  • Muscles / virology*
  • Nucleic Acid Conformation
  • Rad51 Recombinase / genetics*
  • Rad51 Recombinase / metabolism
  • Transduction, Genetic*


  • DNA, Viral
  • DNA-Binding Proteins
  • Muscle Proteins
  • X-ray repair cross complementing protein 3
  • Rad51 Recombinase