Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway.
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.
1] Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA [2] Department of Ophthalmology, University of North Carolina, Chapel Hill, North Carolina, USA.
Matthew L
ML
Hirsch
Chengwen
C
Li
Isabella
I
Bellon
Chaoying
C
Yin
Sai
S
Chavala
Marina
M
Pryadkina
Isabelle
I
Richard
Richard Jude
RJ
Samulski
D000818:Animals; D016466:CHO Cells; D002213:Capsid / Q000378:metabolism*; D003412:Cricetulus; D042003:DNA Packaging; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D004195:Disease Models, Animal; D005123:Eye / Q000821:virology; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D016679:Genome, Viral; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000821:virology*; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D018482:Muscle, Skeletal / Q000821:virology; D009132:Muscles / Q000821:virology*; D009690:Nucleic Acid Conformation; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009124:Muscle Proteins; C110901:X-ray repair cross complementing protein 3; D051135:Rad51 Recombinase