In utero gene transfer is a successful method for transferring genes to the developing fetus. Providing a therapeutic gene to the developing fetus allows for the treatment of a genetic defect before the comorbidities of the disease occur. Many genetic diseases can be detected in utero, and the prenatal treatment of these diseases could prove beneficial. The fetus provides a unique environment for gene transfer because one can influence differentiation and proliferation of target cells. In addition, the fetal environment provides the additional benefit of an immature immune system where vectors are not seen as foreign [1, 2].
This laboratory has shown that in utero gene transfer via amniotic fluid is an effective method for introducing genes into epithelial multipotential stem cells of the lung and intestines [3–8]. Gene transfer is performed at a gestational age comparable to a 5–15 week human (15–16 days in mice; 16–17 days gestation in rats; 90–110 days in rhesus). During this critical time of development, undifferentiated epithelial cells, the targets for the vectors, line the lung and intestine. These cells are the targets for our vectors. Organ development and growth continues after the infection and the differentiated daughters of the targeted cells continue to express the transgene. Short-term adenovirus-mediated in utero gene transfer has now been successfully demonstrated in three species: the mouse, the rat, and the rhesus primate.
The use of recombinant adenovirus is limited to studies examining the role of a specific gene during a very limited time period. The non-integrating properties of the vector limit expression to the lifespan of the cell-approximately 10 to 30 days in the intestine and lung, respectively. Recombinant adeno-associated viruses have the advantage of being maintained for longer periods of time, and the infection of a multipotential stem cell with this vector would have a significant advantage over adenoviral-mediated gene transfer for many disease models. Experiments were performed to determine if rAAV2 would infect the fetal lung and intestines efficiently.
Uptake and expression of AAV2 reporter genes in fetal tissues
Mouse fetuses (20 in two liters) at 15–16 day gestation were infected with rAAV2CMVgfp via intra-amniotic injection of 108 pfu/ml of amniotic fluid. Whole embryos at 72 hours post-therapy were fixed, and frozen sections were prepared for viewing with the deconvoluting microscope. As shown in Fig. 1, the rAAV2 transgene is rapidly transferred and expressed in both the lungs (panel A) and intestines (panel B). All other organs, except for the kidney (see below), were negative for gfp-specific staining.
This laboratory has previously demonstrated that transgenes delivered by in utero gene transfer expressed proteins that were secreted regardless of the presence or absence of a secretory signal [5]. In addition, it was further demonstrated that the kidney, which cannot be infected via intra-amniotic in utero gene transfer, filters virus-delivered transgene products from the circulation. Based on molecular weight, the gap junction in the glomeruli differentially partitions proteins into either the efferent tubules or glomeruli. Proteins greater than 80,000 molecular weight collect in the glomeruli while those less than 80,000 pass through the glomeruli and collect in the efferent tubules. The protein in the efferent tubules appears as an intracellular signal, while in the glomeruli large aggregates of protein were found [5].
By 72 hours post gene transfer, transgene expression was sufficiently high for systemic circulation of protein (Fig. 1, Panel C). Because GFP has a molecular weight below 80,000, it was filtered by the kidneys and collected in the efferent tubules. Although the efferent tubules are positive, there is no evidence of GFP protein in the glomeruli (round clusters of blue-stained nuclei with red-stained membranes. Thus, both adenovirus and rAAV2 vector-delivered transgenes produce high levels of proteins that are released into the systemic circulation and subsequently filtered by the kidney.
Persistence of rAAV2 DNA and transgene expression in rats
To evaluate the expression and long-term persistence of rAAV2 genes and extend the efficacy of in utero rAAV2 to another species, Sprague-Dawley, the rats were treated in utero at 16 days' gestation with the rAAV2CMVgfp virus. Tissues were examined for the presence of gfp-specific protein by immunohistochemistry and DNA by Southern blot analysis.
At nine months of age, rat lungs were harvested and fixed for paraffin sections for immunohistochemistry. As shown in Fig. 2, GFP was readily detected at 9 months of age in the airways of all animals tested. Thus, rAAV2 vectors readily infected the lungs of rats, and transgene expression occurs long-term.
Rats were followed up to one year of age, and DNA was extracted from both the lungs and intestines. Although the intestines are a primary target of in utero gene transfer from the amniotic fluid, no GFP DNA was detected by Southern blot in these tissues. This indicated that the dilution of transgene had decreased to levels below detection and /or that the original transfer efficiency of the intestine multipotential stem cells was poor. In contrast, GFP DNA was found in all the rats' lungs at 12 months of age as demonstrated in Fig. 3.
Non-human primate studies
The in utero uptake of the rAAV2 in the mouse suggests that this vector could be effective, for the reversal of metabolic defects. For this gene therapy to be effective it must translate well from rodents to humans and it must persist over extended periods of time. To test both these parameters a Rhesus macaque was infected with rAAV2-GFP at 100 days' gestation, allowed to deliver, and then tissues examined for GFP expression at 15 months of age (16 months post-infection). This gestation time is comparable to that used for both mice and rats (Figs. 1,2,3; [5]).
The lungs from the NHP showed both type I and type II cells positive for GFP expression (Fig. 4, Panel A). These cells were scattered throughout the lung parenchyma and were readily apparent in all sections of the lung. Thus, rAAV2 DNA and gene expression are maintained in lungs of the NHP.
Like the lungs, the rhesus' intestines showed random (<5%) villi positive for GFP (Fig. 4, Panel B). Because cells in a villi turn over every 72 hours and differentiation occurs outwardly from stem cells located in the crypt, this distribution was consistent with the maintenance of individual clones of stem cells transfected by the rAAV2. In addition, the finding that only random villi were positive for transgene expression was consistent with the rat studies where long-term gfp-specific DNA sequences could not be detected. This suggests that only a small proportion of stem cells in the intestine become infected with the transgene and that maintenance of transgene expression over a long period of time is reduced. In addition, the protein appears to collect in the lacteal, which includes the lymphatics of the intestines.
The finding that transgene protein was present primarily in the lacteal of the intestines suggests that either epithelial cells were secreting the protein into the lymphatic fluid or that phagocytic cells of the lymphatics were scavenging this protein. To determine if the transgene protein was in phagocytic cells or free in the lymphatics, the sections were stained with both fluorescently labeled phalloidin toxins to stain membranes and DAPI for nuclei; and they were then analyzed by high-resolution, deconvoluting microscopy. As shown in Fig. 5, GFP is not associated with specific cells but rather was present in the lymphatic fluid of the intestines. Thus, the secretion of transgene proteins from intestinal epithelial cells results in release into the lymphatics. This finding would account for the protein seen in the kidneys because the lymphatics drain into the general circulation from which proteins would be filtered by the kidneys.