TY - JOUR
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
N1 - Funding Information:
We thank Camille Samulski for reviewing the text and Eric Horowitz for qPCR assistance. This work was supported by the Jain Foundation, the Northwest Genome Engineering Consortium and by the RO1 AI072176 awarded to MLH. NIH grants R01 AI080726, DK084033, AI072176, and U54 AR056953 awarded to RJS also supported this work.
PY - 2013/12
Y1 - 2013/12
N2 - 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.
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.
UR - http://www.scopus.com/inward/record.url?scp=84890119936&partnerID=8YFLogxK
U2 - 10.1038/mt.2013.184
DO - 10.1038/mt.2013.184
M3 - Article
C2 - 23939025
AN - SCOPUS:84890119936
SN - 1525-0016
VL - 21
SP - 2205
EP - 2216
JO - Molecular Therapy
JF - Molecular Therapy
IS - 12
ER -