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

Transfection efficiency of rAAV vectors following in-utero maternal systemic delivery. Pregnant adult dams at E12.5 were treated with 1.3 × 1012 viral genomes rAAV2-CMV-eGFP (n = 1) or 8.4 × 1012 viral genomes rAAV 2/9-CMV-eGFP (n = 1) by tail vein injection. Pups were sacrificed at 2, 4, 6, 8, 10, and 12 weeks of life. Dams were sacrificed at 8 weeks postpartum. Quantitative real-time PCR was performed on DNA isolated from kidneys for viral genome copies. Transfection efficiency is expressed in viral genomes per microgram of extracted DNA. White bars represent mice treated with rAAV2-CMV-eGFP (n = 9) and black bars represent mice treated with rAAV2/9-CMV-eGFP (n = 12). Error bars are mean + SD. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Transfection efficiency of rAAV vectors following in-utero maternal systemic delivery. Pregnant adult dams at E12.5 were treated with 1.3 × 1012 viral genomes rAAV2-CMV-eGFP (n = 1) or 8.4 × 1012 viral genomes rAAV 2/9-CMV-eGFP (n = 1) by tail vein injection. Pups were sacrificed at 2, 4, 6, 8, 10, and 12 weeks of life. Dams were sacrificed at 8 weeks postpartum. Quantitative real-time PCR was performed on DNA isolated from kidneys for viral genome copies. Transfection efficiency is expressed in viral genomes per microgram of extracted DNA. White bars represent mice treated with rAAV2-CMV-eGFP (n = 9) and black bars represent mice treated with rAAV2/9-CMV-eGFP (n = 12). Error bars are mean + SD. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.

Mentions: We quantified the number of viral genomes in renal tissues to determine the viral gene transduction efficiency. At the time of sacrifice, DNA was isolated from kidneys of dams and pups treated with the rAAV2-CMV-eGFP or rAAV2/9-CMV-eGFP vectors or saline-treated controls. Viral genomes were measured by quantitative PCR and compared with a standard curve of GFP plasmids (Figure 4). As expected, maternal tissues contained several orders of magnitude higher viral genomes per microgram DNA (rAAV2/9-CMV-eGFP: 7.61 × 105 and rAAV2-CMV-eGFP: 2.61 × 104) than that found in pups (rAAV2/9-CMV-eGFP: 7.95–2,150 and rAAV2-CMV-eGFP: 57.1–988). Saline-treated dams and pups did not demonstrate any eGFP expression as expected. DNA was also isolated from the kidneys of the second litter of pups for each dam to determine if there was germline transmission of viral vector DNA. No viral genomes could be detected by quantitative PCR in the kidneys of any of the pups of either second litter (data not shown).


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)

Transfection efficiency of rAAV vectors following in-utero maternal systemic delivery. Pregnant adult dams at E12.5 were treated with 1.3 × 1012 viral genomes rAAV2-CMV-eGFP (n = 1) or 8.4 × 1012 viral genomes rAAV 2/9-CMV-eGFP (n = 1) by tail vein injection. Pups were sacrificed at 2, 4, 6, 8, 10, and 12 weeks of life. Dams were sacrificed at 8 weeks postpartum. Quantitative real-time PCR was performed on DNA isolated from kidneys for viral genome copies. Transfection efficiency is expressed in viral genomes per microgram of extracted DNA. White bars represent mice treated with rAAV2-CMV-eGFP (n = 9) and black bars represent mice treated with rAAV2/9-CMV-eGFP (n = 12). Error bars are mean + SD. 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

fig4: Transfection efficiency of rAAV vectors following in-utero maternal systemic delivery. Pregnant adult dams at E12.5 were treated with 1.3 × 1012 viral genomes rAAV2-CMV-eGFP (n = 1) or 8.4 × 1012 viral genomes rAAV 2/9-CMV-eGFP (n = 1) by tail vein injection. Pups were sacrificed at 2, 4, 6, 8, 10, and 12 weeks of life. Dams were sacrificed at 8 weeks postpartum. Quantitative real-time PCR was performed on DNA isolated from kidneys for viral genome copies. Transfection efficiency is expressed in viral genomes per microgram of extracted DNA. White bars represent mice treated with rAAV2-CMV-eGFP (n = 9) and black bars represent mice treated with rAAV2/9-CMV-eGFP (n = 12). Error bars are mean + SD. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
Mentions: We quantified the number of viral genomes in renal tissues to determine the viral gene transduction efficiency. At the time of sacrifice, DNA was isolated from kidneys of dams and pups treated with the rAAV2-CMV-eGFP or rAAV2/9-CMV-eGFP vectors or saline-treated controls. Viral genomes were measured by quantitative PCR and compared with a standard curve of GFP plasmids (Figure 4). As expected, maternal tissues contained several orders of magnitude higher viral genomes per microgram DNA (rAAV2/9-CMV-eGFP: 7.61 × 105 and rAAV2-CMV-eGFP: 2.61 × 104) than that found in pups (rAAV2/9-CMV-eGFP: 7.95–2,150 and rAAV2-CMV-eGFP: 57.1–988). Saline-treated dams and pups did not demonstrate any eGFP expression as expected. DNA was also isolated from the kidneys of the second litter of pups for each dam to determine if there was germline transmission of viral vector DNA. No viral genomes could be detected by quantitative PCR in the kidneys of any of the pups of either second litter (data not shown).

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