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In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates.

Garrett DJ, Larson JE, Dunn D, Marrero L, Cohen JC - BMC Biotechnol. (2003)

Bottom Line: Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates.Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts.Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ochsner Children's Research Institute, Ochsner Clinic Foundation, New Orleans, LA 70121, USA. djgarre@aol.com

ABSTRACT

Background: Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV) have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes.

Methods: Recombinant AAV2 carrying green fluorescent protein (GFP) was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor.

Results: Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year.

Conclusions: Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

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High resolution, three color analysis of GFP protein in NHP intestines. Frozen sections from a 15 month old NHP were stained with the nuclear stain DAPI (blue color) and the actin-specific phalloidin conjugated to fluorescent marker (red color). Three dimensional reconstructions were performed on an automated, deconvoluting microscope. Original magnification 630X
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Figure 5: High resolution, three color analysis of GFP protein in NHP intestines. Frozen sections from a 15 month old NHP were stained with the nuclear stain DAPI (blue color) and the actin-specific phalloidin conjugated to fluorescent marker (red color). Three dimensional reconstructions were performed on an automated, deconvoluting microscope. Original magnification 630X

Mentions: 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.


In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates.

Garrett DJ, Larson JE, Dunn D, Marrero L, Cohen JC - BMC Biotechnol. (2003)

High resolution, three color analysis of GFP protein in NHP intestines. Frozen sections from a 15 month old NHP were stained with the nuclear stain DAPI (blue color) and the actin-specific phalloidin conjugated to fluorescent marker (red color). Three dimensional reconstructions were performed on an automated, deconvoluting microscope. Original magnification 630X
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC239997&req=5

Figure 5: High resolution, three color analysis of GFP protein in NHP intestines. Frozen sections from a 15 month old NHP were stained with the nuclear stain DAPI (blue color) and the actin-specific phalloidin conjugated to fluorescent marker (red color). Three dimensional reconstructions were performed on an automated, deconvoluting microscope. Original magnification 630X
Mentions: 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.

Bottom Line: Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates.Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts.Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ochsner Children's Research Institute, Ochsner Clinic Foundation, New Orleans, LA 70121, USA. djgarre@aol.com

ABSTRACT

Background: Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV) have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes.

Methods: Recombinant AAV2 carrying green fluorescent protein (GFP) was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor.

Results: Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year.

Conclusions: Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

Show MeSH
Related in: MedlinePlus