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. 2010 Feb;21(2):143-8.
doi: 10.1089/hum.2009.126.

Long-term luciferase expression monitored by bioluminescence imaging after adeno-associated virus-mediated fetal gene delivery in rhesus monkeys (Macaca mulatta)

Alice F Tarantal  1 , C Chang I Lee
Affiliations

Long-term luciferase expression monitored by bioluminescence imaging after adeno-associated virus-mediated fetal gene delivery in rhesus monkeys (Macaca mulatta)

Alice F Tarantal et al. Hum Gene Ther. 2010 Feb.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Postnatal bioluminescence imaging (BLI) after prenatal adeno-associated virus (AAV) administration. Fetal rhesus monkeys were administered AAV2/5 (A–F), AAV2/9 (G–L), or AAV2/10 (M–R) by an intrathoracic ultrasound-guided approach. Three animals are shown (one per group) that were imaged with the Xenogen IVIS 200 imaging system after injecting d-luciferin intravenously (100 mg/kg) at 1 month: (A) (1.2 × 109 photons/sec), (G) (7.2 × 109 photons/sec), and (M) (2.4 × 1010 photons/sec); 3 months: (B) (1.0 × 109 photons/sec), (H) (1.9 × 1010 photons/sec), and (N) (7.0 × 1010 photons/sec), 6 months: (C) (1.0 × 109 photons/sec), (I) (1.4 × 1010 photons/sec), and (O) (5.9 × 1010 photons/sec); 12 months: (D) (1.5 × 109 photons/sec), (J) (1.5 × 1010 photons/sec), and (P) (5.6 × 1010 photons/sec), 18 months: (E) (2.2 × 109 photons/sec), (K) (1.6 × 1010 photons/sec), and (Q) (9.2 × 1010 photons/sec), and 24 months: (F) (2.7 × 109 photons/sec), (L) (1.7 × 1010 photons/sec), and (R) (5.0 × 1010 photons/sec). Injections were performed in the right thorax; imaging indicates no evidence of biodistribution to other anatomical sites.
FIG. 2.
FIG. 2.
Postnatal BLI after prenatal intramyocardial AAV administration. Animals administered AAV2/5 (A–C), AAV2/9 (D–F), and AAV2/10 (G–I) are shown at 1 month: (A) (1.3 × 109 photons/sec), (D) (8.3 × 1010 photons/sec), and (G) (3.5 × 109 photons/sec), 3 months: (B) (2.6 × 109 photons/sec), (E) (1.8 × 1011 photons/sec), and (H) (6.5 × 109 photons/sec), and 6 months: (C) (3.3 × 109 photons/sec), (F) (2.1 × 1011 photons/sec), and (I) (1.1 × 1010 photons/sec) postnatal age.
FIG. 3.
FIG. 3.
Three-dimensional reconstruction of bioluminescent images. Three-dimensional reconstruction (A) after intramyocardial administration of AAV vector supernatant was performed, using the diffuse luminescence imaging tomography (DLIT) algorithm inherent in the IVIS 200 imaging system (scale, 1012 photons/sec [shown in black and red]; green plane, transaxial [C]; blue plane, sagittal [D]). The thoracic surface is represented in white. Colors on the surface represent levels of bioluminescence as shown in Fig. 2. Measurements taken from transaxial (C; bar, 8.0 mm) and sagittal (D; bar, 7.6 mm) sections were compared with measures obtained by ultrasound (B; bar, 6.5 mm) and showed an approximate 15% margin of error in the localization of source bioluminescence.
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Comment in

  • A ray of light for fetal therapy.
    Toelen J, Deprest J. Toelen J, et al. Hum Gene Ther. 2010 Feb;21(2):137-9. doi: 10.1089/hum.2010.1015. Hum Gene Ther. 2010. PMID: 20136405 No abstract available.

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