Limits...
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

Immunohistochemistry staining for GFP in kidneys of treated pups and dams. Pregnant adult dams at E12.5 were treated with saline sham (a, c, e, g, i) or 1.0 × 1012 viral genomes rAAV 2/9-NPHS1-eGFP (b, d, f, h, j) by tail vein injection. Pups were sacrificed at 2 (a, b), 4 (c, d), 8 (e, f), and 12 (g, h) weeks of life. (i, j) Dams were sacrificed at 8 weeks postpartum. Kidneys were snap frozen, whole mounted for imaging, cryosectioned (10 μm), and processed for immunohistochemistry. Bars = 200 μm. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362350&req=5

fig5: Immunohistochemistry staining for GFP in kidneys of treated pups and dams. Pregnant adult dams at E12.5 were treated with saline sham (a, c, e, g, i) or 1.0 × 1012 viral genomes rAAV 2/9-NPHS1-eGFP (b, d, f, h, j) by tail vein injection. Pups were sacrificed at 2 (a, b), 4 (c, d), 8 (e, f), and 12 (g, h) weeks of life. (i, j) Dams were sacrificed at 8 weeks postpartum. Kidneys were snap frozen, whole mounted for imaging, cryosectioned (10 μm), and processed for immunohistochemistry. Bars = 200 μm. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.

Mentions: When kidneys of pups treated with rAAV 2/9-NPHS1-eGFP were examined by IH, significant staining of renal glomeruli was observed compared with sham-injected pups at 2, 4, 8, and 12 weeks of life (Figure 5a–h). More robust staining was identified in treated dams compared with the pups as shown in Figure 5i–j. When analyzed by IF with costaining with WT-1 and DAPI (Figure 6a–c), similar staining of the glomeruli was identified that was more robust in the dams and absent in saline-injected controls.


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)

Immunohistochemistry staining for GFP in kidneys of treated pups and dams. Pregnant adult dams at E12.5 were treated with saline sham (a, c, e, g, i) or 1.0 × 1012 viral genomes rAAV 2/9-NPHS1-eGFP (b, d, f, h, j) by tail vein injection. Pups were sacrificed at 2 (a, b), 4 (c, d), 8 (e, f), and 12 (g, h) weeks of life. (i, j) Dams were sacrificed at 8 weeks postpartum. Kidneys were snap frozen, whole mounted for imaging, cryosectioned (10 μm), and processed for immunohistochemistry. Bars = 200 μm. 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

fig5: Immunohistochemistry staining for GFP in kidneys of treated pups and dams. Pregnant adult dams at E12.5 were treated with saline sham (a, c, e, g, i) or 1.0 × 1012 viral genomes rAAV 2/9-NPHS1-eGFP (b, d, f, h, j) by tail vein injection. Pups were sacrificed at 2 (a, b), 4 (c, d), 8 (e, f), and 12 (g, h) weeks of life. (i, j) Dams were sacrificed at 8 weeks postpartum. Kidneys were snap frozen, whole mounted for imaging, cryosectioned (10 μm), and processed for immunohistochemistry. Bars = 200 μm. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
Mentions: When kidneys of pups treated with rAAV 2/9-NPHS1-eGFP were examined by IH, significant staining of renal glomeruli was observed compared with sham-injected pups at 2, 4, 8, and 12 weeks of life (Figure 5a–h). More robust staining was identified in treated dams compared with the pups as shown in Figure 5i–j. When analyzed by IF with costaining with WT-1 and DAPI (Figure 6a–c), similar staining of the glomeruli was identified that was more robust in the dams and absent in saline-injected controls.

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