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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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rAAV2 mediated gene transfer in the mouse fetus. Fetuses at 15–16 day gestation were infected with rAAV2CMVgfp via intra-amniotic transfer. At 72 hours post-infection whole fetuses were fixed and section for deconvoluting microscopic analysis. Sections were stained with the nuclear stain DAPI (blue) and phalloidin (red) staining of actin. A-lung; B-intestines; and C-kidney. Original magnification 630X
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Figure 1: rAAV2 mediated gene transfer in the mouse fetus. Fetuses at 15–16 day gestation were infected with rAAV2CMVgfp via intra-amniotic transfer. At 72 hours post-infection whole fetuses were fixed and section for deconvoluting microscopic analysis. Sections were stained with the nuclear stain DAPI (blue) and phalloidin (red) staining of actin. A-lung; B-intestines; and C-kidney. Original magnification 630X

Mentions: 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.


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)

rAAV2 mediated gene transfer in the mouse fetus. Fetuses at 15–16 day gestation were infected with rAAV2CMVgfp via intra-amniotic transfer. At 72 hours post-infection whole fetuses were fixed and section for deconvoluting microscopic analysis. Sections were stained with the nuclear stain DAPI (blue) and phalloidin (red) staining of actin. 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 1: rAAV2 mediated gene transfer in the mouse fetus. Fetuses at 15–16 day gestation were infected with rAAV2CMVgfp via intra-amniotic transfer. At 72 hours post-infection whole fetuses were fixed and section for deconvoluting microscopic analysis. Sections were stained with the nuclear stain DAPI (blue) and phalloidin (red) staining of actin. A-lung; B-intestines; and C-kidney. Original magnification 630X
Mentions: 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.

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