AAV-mediated gene targeting is significantly enhanced by transient inhibition of nonhomologous end joining or the proteasome in vivo

Abstract

Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah(-/-)Ku70(-/-) double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy.

FIG. 1.

Transient non-homologous end joining (NHEJ) or proteasome inhibition enhances gene targeting. Frequencies of corrected nodules were quantified by counting the number of FAH+ clones per x hepatocytes (1/x) from adult mice harvested 4 weeks posttreatment. Mean and SD are shown, with the number of independent animals analyzed above each bar. Black, adeno-associated virus (AAV); white, AAV + vanillin; gray, AAV + bortezomib; lines, AAV + vanillin + bortezomib.

FIG. 2.

Liver immunohistochemistry. (a) Fumarylacetoacetate hydrolase positive (FAH+) staining from adult Fah^+/+ control mouse; (b) FAH− staining from adult Fah^−/− control mouse; (c) FAH stain on adult Fah^−/− mouse treated with AAV, (d) AAV + vanillin, (e) AAV + bortezomib, or (f) AAV + vanillin + bortezomib; (g) hematoxylin and eosin (H&E) stain on adult Fah^−/− mouse treated with vanillin; (h) H&E stain on adult Fah^−/− mouse treated with bortezomib; (i) FAH stain on adult Fah^−/− serial transplant recipients; (j) FAH stain on Fah^−/−Ku70^+/+ neonate treated with AAV; (k) FAH stain on Fah^−/−Ku70^+/− neonate treated with AAV; (l) FAH stain on Fah^−/−Ku70^−/− neonate treated with AAV. Scale bar=10 μm.

FIG. 3.

Sexually dimorphic responses to AAV and vanillin. Frequencies of corrected nodules were quantified by counting the number of FAH+ clones per x hepatocytes (1/x) from neonates harvested at 3 weeks and adults harvested 4 weeks posttreatment. Mean and SD are shown, with the number of independent animals analyzed above each bar. Black, AAV; white, AAV + vanillin; M, male; F, female.

FIG. 4.

NHEJ inhibition is responsible for increases in gene targeting seen with vanillin. Frequencies of corrected nodules were quantified by counting the number of FAH+ clones per x hepatocytes (1/x) from neonates harvested at 3 weeks posttreatment. Mean and SD are shown, with the number of independent animals analyzed above each bar. Black, Fah^−/−Ku70^+/+; white, Fah^−/−Ku70^+/−; gray, Fah^−/−Ku70^−/−.