Long-term luciferase expression monitored by bioluminescence imaging after adeno-associated virus-mediated fetal gene delivery in rhesus monkeys (Macaca mulatta).
The safety and efficiency of fetal adeno-associated virus (AAV) gene delivery in rhesus monkeys and long-term monitoring of transgene expression by bioluminescence imaging (BLI) were evaluated. Early second-trimester fetal monkeys were administered AAV2/5, AAV2/9, or AAV2/10 vector supernatant preparations expressing firefly luciferase under the control of the cytomegalovirus promoter, using an intrathoracic (n = 6) or intramyocardial (n = 6) approach and established ultrasound-guided techniques. Postnatal BLI was performed monthly up to 6 months postnatal age (n = 12) and then every 3 months thereafter to monitor transgene expression up to 24 months postnatal age (27 months after gene transfer; n = 6). All AAV serotypes showed greater than 1.0 x 10(9) photons/sec at all time points evaluated with limited biodistribution to nontargeted anatomical sites. The highest levels of bioluminescence (photons per second) observed were noted with AAV2/9 and AAV2/10 when the three vector constructs were compared. To correlate in vivo findings at the tissue level, specimens were collected from selected animals and analyzed. Three-dimensional reconstruction showed that firefly luciferase expression was consistent with imaging and morphometric measures. These findings suggest that (1) high levels of AAV-mediated firefly luciferase expression can be found after fetal AAV gene transfer and without any evidence of adverse effects; (2) the intercostal muscles, myocardium, and muscular component of the diaphragm of developing fetuses are readily transduced with AAV2/5, AAV2/9, or AAV2/10; and (3) postnatal outcomes and long-term luciferase expression can be effectively monitored by BLI in young rhesus monkeys.
Tarantal
Alice F
AF
Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases, California National Primate Research Center, Davis, CA 95616, USA. aftarantal@primate.ucdavis.edu
Lee
C Chang I
CC
D000818:Animals; D000831:Animals, Newborn / Q000235:genetics / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005260:Female; D046128:Fetal Therapies / Q000379:methods*; D005333:Fetus / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008253:Macaca mulatta / Q000196:embryology / Q000235:genetics; D011247:Pregnancy; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics
D008164:Luminescent Proteins; D049409:Luciferases, Firefly