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Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9.

Picconi JL, Muff-Luett MA, Wu D, Bunchman E, Schaefer F, Brophy PD - Mol Ther Methods Clin Dev (2014)

Bottom Line: Gene therapy targeting of kidneys has been largely unsuccessful.An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life.Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics , Iowa City, Iowa, USA ; Department of Pediatrics, University of Iowa Children's Hospital , Iowa City, Iowa, USA.

ABSTRACT
Gene therapy targeting of kidneys has been largely unsuccessful. Recently, a recombinant adeno-associated virus (rAAV) vector was used to target adult mouse kidneys. Our hypothesis is that a pseudotyped rAAV 2/9 vector can produce fetal kidney-specific expression of the green fluorescent protein (GFP) gene following maternal tail vein injection of pregnant mice. Pregnant mice were treated with rAAV2/9 vectors with either the ubiquitous cytomegalovirus promoter or the minimal NPHS1 promoter to drive kidney-specific expression of GFP. Kidneys from dams and pups were analyzed for vector DNA, gene expression, and protein. Vector DNA was identified in kidney tissue out to 12 weeks at low but stable levels, with levels higher in dams than that in pups. Robust GFP expression was identified in the kidneys of both dams and pups treated with the cytomegalovirus (CMV)-enhanced green fluorescent protein (eGFP) vector. When treated with the NPHS1-eGFP vector, dams and pups showed expression of GFP only in kidneys, localized to the glomeruli. An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life. Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence staining for GFP in adult mouse kidneys. Adult mice (n = 2) were treated with 1 × 1012 viral genomes rAAV 2/9-CMV-eGFP by tail vein injection. Eight weeks postinjection, kidneys were snap frozen and whole mounted for imaging, then cryosectioned (10 μm) and processed for immunofluorescence staining. Bars = 2 mm (a) and 500 μm (b). CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
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fig1: Immunofluorescence staining for GFP in adult mouse kidneys. Adult mice (n = 2) were treated with 1 × 1012 viral genomes rAAV 2/9-CMV-eGFP by tail vein injection. Eight weeks postinjection, kidneys were snap frozen and whole mounted for imaging, then cryosectioned (10 μm) and processed for immunofluorescence staining. Bars = 2 mm (a) and 500 μm (b). CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.

Mentions: Prior to undertaking the in-utero administration of the rAAV 2/9-cytomegalovirus (CMV)-enhanced green fluorescent protein (eGFP) vector, we sought to replicate previous reports of AAV9 affinity for kidney tissue. Following tail vein injection of the rAAV 2/9-CMV-eGFP vector to adult mice, immunofluorescence (IF) and immunohistochemistry (IH) staining of harvested tissues for GFP showed consistent staining of all tissues analyzed (adrenal, bladder, brain, heart, kidney, liver, lung, ovary, pancreas, salivary gland, skeletal muscle, spleen, testis, thymus, thyroid, and uterus). Specifically, the AAV2/9-CMV-eGFP vector showed significant tropism for the kidney, localizing to the glomeruli and tubules (Figure 1a,b).


Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9.

Picconi JL, Muff-Luett MA, Wu D, Bunchman E, Schaefer F, Brophy PD - Mol Ther Methods Clin Dev (2014)

Immunofluorescence staining for GFP in adult mouse kidneys. Adult mice (n = 2) were treated with 1 × 1012 viral genomes rAAV 2/9-CMV-eGFP by tail vein injection. Eight weeks postinjection, kidneys were snap frozen and whole mounted for imaging, then cryosectioned (10 μm) and processed for immunofluorescence staining. Bars = 2 mm (a) and 500 μm (b). CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362350&req=5

fig1: Immunofluorescence staining for GFP in adult mouse kidneys. Adult mice (n = 2) were treated with 1 × 1012 viral genomes rAAV 2/9-CMV-eGFP by tail vein injection. Eight weeks postinjection, kidneys were snap frozen and whole mounted for imaging, then cryosectioned (10 μm) and processed for immunofluorescence staining. Bars = 2 mm (a) and 500 μm (b). CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
Mentions: Prior to undertaking the in-utero administration of the rAAV 2/9-cytomegalovirus (CMV)-enhanced green fluorescent protein (eGFP) vector, we sought to replicate previous reports of AAV9 affinity for kidney tissue. Following tail vein injection of the rAAV 2/9-CMV-eGFP vector to adult mice, immunofluorescence (IF) and immunohistochemistry (IH) staining of harvested tissues for GFP showed consistent staining of all tissues analyzed (adrenal, bladder, brain, heart, kidney, liver, lung, ovary, pancreas, salivary gland, skeletal muscle, spleen, testis, thymus, thyroid, and uterus). Specifically, the AAV2/9-CMV-eGFP vector showed significant tropism for the kidney, localizing to the glomeruli and tubules (Figure 1a,b).

Bottom Line: Gene therapy targeting of kidneys has been largely unsuccessful.An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life.Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics , Iowa City, Iowa, USA ; Department of Pediatrics, University of Iowa Children's Hospital , Iowa City, Iowa, USA.

ABSTRACT
Gene therapy targeting of kidneys has been largely unsuccessful. Recently, a recombinant adeno-associated virus (rAAV) vector was used to target adult mouse kidneys. Our hypothesis is that a pseudotyped rAAV 2/9 vector can produce fetal kidney-specific expression of the green fluorescent protein (GFP) gene following maternal tail vein injection of pregnant mice. Pregnant mice were treated with rAAV2/9 vectors with either the ubiquitous cytomegalovirus promoter or the minimal NPHS1 promoter to drive kidney-specific expression of GFP. Kidneys from dams and pups were analyzed for vector DNA, gene expression, and protein. Vector DNA was identified in kidney tissue out to 12 weeks at low but stable levels, with levels higher in dams than that in pups. Robust GFP expression was identified in the kidneys of both dams and pups treated with the cytomegalovirus (CMV)-enhanced green fluorescent protein (eGFP) vector. When treated with the NPHS1-eGFP vector, dams and pups showed expression of GFP only in kidneys, localized to the glomeruli. An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life. Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

No MeSH data available.


Related in: MedlinePlus