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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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Related in: MedlinePlus

Persistence and expression of transgenes following in utero rAAV2 gene therapy. In utero gene therapy withrAAV2CMVgfp was performed in NHP and the tissues analyzed for GFP expression at 15 months of age. All sections were stained with the nuclear-specific DAPI (blue color) and visualized on a deconvoluting microscope. A – Lung; B – Intestines; and C – Kidney. Original magnification 630X
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Figure 4: Persistence and expression of transgenes following in utero rAAV2 gene therapy. In utero gene therapy withrAAV2CMVgfp was performed in NHP and the tissues analyzed for GFP expression at 15 months of age. All sections were stained with the nuclear-specific DAPI (blue color) and visualized on a deconvoluting microscope. A – Lung; B – Intestines; and C – Kidney. Original magnification 630X

Mentions: 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.


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)

Persistence and expression of transgenes following in utero rAAV2 gene therapy. In utero gene therapy withrAAV2CMVgfp was performed in NHP and the tissues analyzed for GFP expression at 15 months of age. All sections were stained with the nuclear-specific DAPI (blue color) and visualized on a deconvoluting microscope. A – Lung; B – Intestines; and C – Kidney. Original magnification 630X
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Persistence and expression of transgenes following in utero rAAV2 gene therapy. In utero gene therapy withrAAV2CMVgfp was performed in NHP and the tissues analyzed for GFP expression at 15 months of age. All sections were stained with the nuclear-specific DAPI (blue color) and visualized on a deconvoluting microscope. A – Lung; B – Intestines; and C – Kidney. Original magnification 630X
Mentions: 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.

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