Bioluminescent virion shells: new tools for quantitation of AAV vector dynamics in cells and live animals.
Current technologies for visualizing infectious pathways of viruses rely on fluorescent labeling of capsid proteins by chemical conjugation or genetic manipulation. For noninvasive in vivo imaging of such agents in mammalian tissue, we engineered bioluminescent Gaussia luciferase-tagged Adeno-associated viral (gLuc/AAV) vectors. The enzyme was incorporated into recombinant AAV serotypes 1, 2 and 8 capsids by fusing to the N-terminus of the VP2 capsid subunit to yield bioluminescent virion shells. The gLuc/AAV vectors were used to quantify kinetics of cell-surface-binding by AAV2 capsids in vitro. Bioluminescent virion shells displayed an exponential decrease in luminescent signal following cellular uptake in vitro. A similar trend was observed following intramuscular injection in vivo, although the rate of decline in bioluminescent signal varied markedly between AAV serotypes. gLuc/AAV1 and gLuc/AAV8 vectors displayed rapid decrease in bioluminescent signal to background levels within 30 min, whereas the signal from gLuc/AAV2 vectors persisted for over 2 h. Bioluminescent virion shells might be particularly useful in quantifying dynamics of viral vector uptake in cells and peripheral tissues in live animals.
Asokan
A
A
Department of Pharmacology, Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7352, USA.
Johnson
J S
JS
Li
C
C
Samulski
R J
RJ
D000818:Animals; D002213:Capsid / Q000378:metabolism; D004274:DNA, Recombinant / Q000008:administration & dosage / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006614:Hindlimb; D006801:Humans; D007273:Injections, Intramuscular; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D014771:Virion / Q000235:genetics* / Q000378:metabolism; D019065:Virus Assembly
D004274:DNA, Recombinant; D049452:Green Fluorescent Proteins; D008156:Luciferases