Lentiviral Vector Gene Transfer into Fetal Rhesus Monkeys (Macaca mulatta): Lung-Targeting Approaches

Abstract

We previously reported the efficiency of gene transfer in fetal monkeys using retroviral vectors and an intraperitoneal (IP) approach. Here, we explored intrapulmonary administration to determine whether gene transfer can be limited to the developing lung. The HIV-1-derived lentiviral vector (VSV-G pseudotyped; 1 x 10(7) infectious particles/fetus), using the enhanced green fluorescent protein (EGFP) as a reporter, was directly injected into fetal lung with ultrasound guidance (n=4; 55 or 70 days gestation; term 165+/-10 days). Fetuses were monitored sonographically, fetal/maternal blood samples collected during gestation, and four of four healthy newborns were delivered at term. All lung lobes were positive for the transgene (< or = 1%) when assessed by PCR, and transgene expression was observed by direct fluorescence microscopy and flow cytometry. The results of this study show the following: (1) successful gene transfer in fetal monkeys using an intrapulmonary approach; (2) less transduction of non-pulmonary tissues with gene transfer at 70 days gestation compared with 55 days gestation or use of an IP approach; (3) that the pulmonary epithelium was EGFP-positive by immunohistochemistry; and (4) no evidence of transplacental transport of vector sequences or antibody responses in the dams. The results of these investigations indicate the efficiency of fetal gene transfer by intrapulmonary delivery, and emphasize the importance of the fetal monkey as a preclinical model system for exploring in utero genetic treatment strategies for pulmonary disorders.

HIV-1-derived lentiviral vectors and fetal route of administration on transgene biodistribution and expression in rhesus monkeys

“…Previous studies in nonhuman primates support the view that direct fetal injection of viral vectors is an effective method for obtaining high levels of persistent gene transduction in a wide array of tissues. 5,6 In the current study, we have utilized quantitative real-time PCR assays to calculate the level of lentiviral transduction and the EGFP mRNA transcripts in transduced tissues. This approach allows one to make direct comparisons between animals under a variety of experimental conditions such as gene transfer at different gestational ages, different routes of administration, and different vector constructs.…”

“…In these tissues, the level of gene marking was in the range of 3-to 2200-fold higher compared to other organs; in addition, the levels of mRNA EGFP transcripts were in the range of 11-to 680-fold higher (Figure 5a and b) ( Table 5). In nonabdominal organs, the level of gene transduction was on average seven-fold (range [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] higher in the animals injected at 40 versus 60 days gestation. No mRNA EGFP transcripts were found in nonabdominal organs of the animals injected at 60 days gestation (Figure 5b).…”

“…5,6 Total body weights and measures (hand, foot, humerus, and femur lengths; biparietal and occipitofrontal diameters; head, arm, and chest circumferences; crownrump lengths) were assessed, then all organs were removed and weighed after carefully examining the thoracic and abdominal cavities, and included the cerebrum, lung, thorax (intercostal muscles), trachea, esophagus, heart, pericardium, thymus, spleen, liver, axillary and mesenteric lymph nodes, pancreas, adrenals, kidneys, gonads, gastrointestinal tract, diaphragm, omentum, peritoneum, and bone marrow. All tissues collected for PCR were placed in 1.5 ml microcentrifuge tubes and immediately frozen in liquid nitrogen and stored at pÀ801C.…”

“…5,6 PBMCs were obtained from blood samples collected into EDTA. Briefly, 1-3 ml of blood were obtained from a peripheral vessel (volume dependent on age) or from the umbilical cord at birth, and placed into vacutainer tubes with PBMCs were resuspended in selection buffer (0.5% BSA and 2 mM EDTA in PBS) and incubated for 10 min at 41C with anti-CD32 (nonspecific blocking immunoglobulin to FcRIIA), followed by a 20 min incubation at 41C with anti-CD34 monoclonal antibody (clone 563; StemCell Technologies, Vancouver, BC, Canada).…”

“…Several studies have demonstrated efficient gene transfer using lentiviral vectors in a variety of animal models [2][3][4] including non-human primates. 5,6 Lentiviral vectors can efficiently mediate gene transfer in a wide range of dividing and nondividing quiescent cells, 7 and maintain gene expression for extended periods of time. Differences in the transcriptional pattern in transduced tissues that may occur under varied experimental conditions include the type of viral construct used, the viral titer transferred, and the timing of fetal gene delivery, all of which can help to determine the best conditions for future human application.…”