Efficient gene transfer into the mouse lung by fetal intratracheal injection of rAAV2/6.2

Abstract

Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for β-galactosidase (β-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.

Figure 1

Comparison of the efficiency of intrapulmonary delivery of fluorescent microspheres by intratracheal (i.t.) versus intra-amniotic (i.a.) injection. (a,b) Examination of whole-body distribution of fluorescent microspheres 24 hours after i.t. or i.a. injection. c,e show the presence of fluorescent microspheres in the fetal lung after i.t. and i.a. injection, respectively. Fluorescent microspheres are also present in the oral (white arrow) and nasal (yellow arrow) cavity of the i.t.-injected fetus (a) and in a lower amount in the i.a.-injected fetus (b). (d,f) The gastrointestinal tract is positive for both the i.t.- and the i.a.-injected animal. a,b, Bar = 2 mm; c–f, bar = 200 µm.

Figure 2

Bioluminescence imaging (BLI) of transgene expression after rAAV2/6.2-mediated gene delivery in fetal lung. (a,b) Low-dose rAAV2/6.2 vectors encoding β-gal [2 × 10⁹ genome copies (GC)/fetus] or fLuc (2 × 10⁸ GC/fetus) were co-injected i.t. or i.a. in fetal Naval Medical Research Institute mice at E18 and followed up by noninvasive BLI at 1 and 4 weeks after injection. (c) Quantification of total photon flux after low-dose rAAV2/6.2. (d,e) BLI signal at 1 and 4 weeks after injection after high-titer rAAV2/6.2 (3 × 10¹⁰ GC/fetus for β-gal and fLuc) administration with corresponding quantification of (f) total photon flux. All animals were scanned, separated by black partitions, to avoid scattering of photons to neighboring animals. The pseudocolor scale depicts the photon flux per second, per square centimeter per steradian (p/s/cm²/sr). Measurements were obtained in a 4.3 cm² rectangular region of interest. Please note that the scales of the BL images are different between the time points. Mean ± SD, analysis of variance, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. neg., negative.

Figure 3

Combination of bioluminescence imaging (BLI) and magnetic resonance imaging (MRI) for accurate localization of gene expression in vivo. Combined BL-MR images of i.t.-injected animals were acquired at week 1. (a,b) BL imaging revealed a signal emanating from the neck and the upper thoracic region. (c,d) Co-registration of MRI with BLI showed that luciferase gene expression was localized in the pulmonary region after a (e) correct injection but in neck and abdominal area after an (f) incorrect injection. X-gal staining confirmed that gene expression was situated in the (g) conducting airways and (i) alveoli of the murine lung after a correct i.t. injection and in the (h) paratracheal space and (j) liver parenchyma after an incorrect i.t. injection. g,i, Bar = 25 µm; h,j, bar = 100 µm.

Figure 4

Transduction efficiency of rAAV2/6.2 in murine airway epithelium after fetal intratracheal injection. (a,b) Quantification of the percentage of transduced cells in conducting airways and alveoli after fetal i.t. injection of low (2 × 10⁹ GC/fetus) and high titer (3 × 10¹⁰ GC/fetus) rAAV2/6.2, respectively. Transduction efficiency of conducting airways was determined by counting the total number of β-gal positive cells in the large airways (trachea and mainstem bronchi) relative to the total number of epithelial cells present on three cross-sections spaced 200 µm apart at ×20 magnification. Alveolar cell transduction was estimated in three cross-sections spaced 200 µm apart using a 400 × 400 µm counting frame at ×20 magnification to systematically sample the lung parenchyma of the entire lung cross-section. To determine the percentage of β-gal positive alveolar cells, the ratio of the average number of β-gal positive cells to the average cell density was made per counting frame. Transduction levels are compared between 1 and 4 weeks after vector administration. (c) Transduction efficiency of the conducting airways was further subdivided and quantified in the trachea, bronchi, and bronchioles, respectively, at 1 and 4 weeks after fetal i.t. injection of high-titer rAAV2/6.2. Mean ± SD, analysis of variance, Student's t-test, *P < 0.05.

Figure 5

rAAV2/6.2-mediated transgene expression in murine airway epithelium after fetal intratracheal injection. β-gal gene transfer to the fetal mouse lung was assessed by X-gal staining. Sections were counterstained with paracarmine. Transduction efficiency is illustrated after high-dose rAAV2/6.2 vector administration (3 × 10¹⁰ GC/fetus) at weeks 1 and 4. Representative images of different lung regions are given at 1 and 4 weeks after fetal i.t. injection, respectively, showing the trachea, the bronchi, the bronchioles, and the alveoli. A high magnification image depicts β-gal positive ciliated (bottom left image) and alveolar cells (bottom middle image) at week 1. Absence of reporter gene expression in a control lung (bottom right image). Bar = 50 µm.