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piggyBac-mediated phenotypic correction of factor VIII deficiency.

Staber JM, Pollpeter MJ, Arensdorf A, Sinn PL, Rutkowski DT, McCray PB - Mol Ther Methods Clin Dev (2014)

Bottom Line: In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity.To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress.These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.

View Article: PubMed Central - PubMed

Affiliation: Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA ; Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA.

ABSTRACT
Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB), to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.

No MeSH data available.


Related in: MedlinePlus

Phenotypic correction of hemophilia A in a mouse model. At 16 or 24 weeks after injection (time of sacrifice), functional correction was observed in mice treated with the PB-coFVIII-BDD transposon and the hyperactive transposase. LR, Lactated Ringer’s treated FVIII  mice; coFVIII-BDD, mice treated with PB-coFVIII-BDD + iPB7; WT, wild-type mice; Inactive, mice treated with PB-coFVIII-BDD + inactive transposase. (a) Blood loss was quantified by measuring the weight of blood collected in normal saline. (b) Hemoglobin was quantified via absorbance. Columns indicate means ± SE. *P < 0.0001 measured via a one-way analysis of variance. **P < 0.05 measured via t-test. ns indicates P > 0.05.
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fig5: Phenotypic correction of hemophilia A in a mouse model. At 16 or 24 weeks after injection (time of sacrifice), functional correction was observed in mice treated with the PB-coFVIII-BDD transposon and the hyperactive transposase. LR, Lactated Ringer’s treated FVIII mice; coFVIII-BDD, mice treated with PB-coFVIII-BDD + iPB7; WT, wild-type mice; Inactive, mice treated with PB-coFVIII-BDD + inactive transposase. (a) Blood loss was quantified by measuring the weight of blood collected in normal saline. (b) Hemoglobin was quantified via absorbance. Columns indicate means ± SE. *P < 0.0001 measured via a one-way analysis of variance. **P < 0.05 measured via t-test. ns indicates P > 0.05.

Mentions: Hemophilia A mice demonstrate significant bleeding after a tail clip.17 To evaluate functional correction of the bleeding phenotype, blood loss following a tail clip was measured at the time of sacrifice. Thirteen of sixteen mice receiving the PB-coFVIII-BDD transposon + iPB7 and without inhibitors revealed partial to full correction as determined by two metrics (Figure 5). Total blood loss was significantly less in mice receiving the PB-coFVIII BDD vector + iPB7 (n = 16) compared to LR-treated (n = 8) or inactive-transposase-treated (n = 5) hemophilia A littermate mice (P < 0.05). Mice demonstrating FVIII antibodies (n = 3) had similar results to LR-treated mice demonstrating the neutralizing effect of the inhibitors. There was no significant difference between wild-type mice and mice receiving the PB-coFVIII-BDD transposon + iPB7 without inhibitors. Therefore, the PB transposon vector system conferred partial to full correction of the bleeding phenotype in most animals.


piggyBac-mediated phenotypic correction of factor VIII deficiency.

Staber JM, Pollpeter MJ, Arensdorf A, Sinn PL, Rutkowski DT, McCray PB - Mol Ther Methods Clin Dev (2014)

Phenotypic correction of hemophilia A in a mouse model. At 16 or 24 weeks after injection (time of sacrifice), functional correction was observed in mice treated with the PB-coFVIII-BDD transposon and the hyperactive transposase. LR, Lactated Ringer’s treated FVIII  mice; coFVIII-BDD, mice treated with PB-coFVIII-BDD + iPB7; WT, wild-type mice; Inactive, mice treated with PB-coFVIII-BDD + inactive transposase. (a) Blood loss was quantified by measuring the weight of blood collected in normal saline. (b) Hemoglobin was quantified via absorbance. Columns indicate means ± SE. *P < 0.0001 measured via a one-way analysis of variance. **P < 0.05 measured via t-test. ns indicates P > 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362371&req=5

fig5: Phenotypic correction of hemophilia A in a mouse model. At 16 or 24 weeks after injection (time of sacrifice), functional correction was observed in mice treated with the PB-coFVIII-BDD transposon and the hyperactive transposase. LR, Lactated Ringer’s treated FVIII mice; coFVIII-BDD, mice treated with PB-coFVIII-BDD + iPB7; WT, wild-type mice; Inactive, mice treated with PB-coFVIII-BDD + inactive transposase. (a) Blood loss was quantified by measuring the weight of blood collected in normal saline. (b) Hemoglobin was quantified via absorbance. Columns indicate means ± SE. *P < 0.0001 measured via a one-way analysis of variance. **P < 0.05 measured via t-test. ns indicates P > 0.05.
Mentions: Hemophilia A mice demonstrate significant bleeding after a tail clip.17 To evaluate functional correction of the bleeding phenotype, blood loss following a tail clip was measured at the time of sacrifice. Thirteen of sixteen mice receiving the PB-coFVIII-BDD transposon + iPB7 and without inhibitors revealed partial to full correction as determined by two metrics (Figure 5). Total blood loss was significantly less in mice receiving the PB-coFVIII BDD vector + iPB7 (n = 16) compared to LR-treated (n = 8) or inactive-transposase-treated (n = 5) hemophilia A littermate mice (P < 0.05). Mice demonstrating FVIII antibodies (n = 3) had similar results to LR-treated mice demonstrating the neutralizing effect of the inhibitors. There was no significant difference between wild-type mice and mice receiving the PB-coFVIII-BDD transposon + iPB7 without inhibitors. Therefore, the PB transposon vector system conferred partial to full correction of the bleeding phenotype in most animals.

Bottom Line: In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity.To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress.These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.

View Article: PubMed Central - PubMed

Affiliation: Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA ; Stead Family Department of Pediatrics, Carver College of Medicine, University of Iowa , Iowa City, Iowa, USA.

ABSTRACT
Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB), to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.

No MeSH data available.


Related in: MedlinePlus