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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

No evidence of liver ER stress. At 16 (n = 4) or 24 (n = 5) weeks after injection (time of sacrifice), liver tissue was assessed for signs of the UPR and ER stress. Gene expression levels for (a) BiP, (b) CHOP, and (c) EDEM were assessed by qPCR. PB-coFVIII indicates mice treated with PB-coFVIII-BDD transposon and iPB7 (n = 9 total). Positive control mice (n = 3) received tunicamycin. FVIII  animals represent both C57 and 129SVJ/B6 backgrounds (n = 3 each). Vector alone group received PB transposon without the FVIII transgene (n = 4). Columns indicate means ± SE.
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fig4: No evidence of liver ER stress. At 16 (n = 4) or 24 (n = 5) weeks after injection (time of sacrifice), liver tissue was assessed for signs of the UPR and ER stress. Gene expression levels for (a) BiP, (b) CHOP, and (c) EDEM were assessed by qPCR. PB-coFVIII indicates mice treated with PB-coFVIII-BDD transposon and iPB7 (n = 9 total). Positive control mice (n = 3) received tunicamycin. FVIII animals represent both C57 and 129SVJ/B6 backgrounds (n = 3 each). Vector alone group received PB transposon without the FVIII transgene (n = 4). Columns indicate means ± SE.

Mentions: Previous studies in mice suggest that short term expression of human BDD FVIII results in hepatocyte stress following plasmid-mediated gene transfer.19 We therefore asked whether short- or long-term FVIII expression via PB induced this cellular pathology. ER stress markers, including binding immunoglobulin protein (BiP), the UPR-regulated CCAAT/enhancer-binding protein homologous protein (CHOP), and ER degradation-enhancing α-mannosidase-like protein (EDEM), are elevated after initiation of the unfolded protein response (UPR).19–21 In comparing the abundance of these transcripts at 1 day and 16–24 weeks after gene transfer, we found no evidence of stress in animals receiving PB-coFVIII-BDD and no significant difference between experimental groups and sham controls at either time point (Figure 4).


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)

No evidence of liver ER stress. At 16 (n = 4) or 24 (n = 5) weeks after injection (time of sacrifice), liver tissue was assessed for signs of the UPR and ER stress. Gene expression levels for (a) BiP, (b) CHOP, and (c) EDEM were assessed by qPCR. PB-coFVIII indicates mice treated with PB-coFVIII-BDD transposon and iPB7 (n = 9 total). Positive control mice (n = 3) received tunicamycin. FVIII  animals represent both C57 and 129SVJ/B6 backgrounds (n = 3 each). Vector alone group received PB transposon without the FVIII transgene (n = 4). Columns indicate means ± SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: No evidence of liver ER stress. At 16 (n = 4) or 24 (n = 5) weeks after injection (time of sacrifice), liver tissue was assessed for signs of the UPR and ER stress. Gene expression levels for (a) BiP, (b) CHOP, and (c) EDEM were assessed by qPCR. PB-coFVIII indicates mice treated with PB-coFVIII-BDD transposon and iPB7 (n = 9 total). Positive control mice (n = 3) received tunicamycin. FVIII animals represent both C57 and 129SVJ/B6 backgrounds (n = 3 each). Vector alone group received PB transposon without the FVIII transgene (n = 4). Columns indicate means ± SE.
Mentions: Previous studies in mice suggest that short term expression of human BDD FVIII results in hepatocyte stress following plasmid-mediated gene transfer.19 We therefore asked whether short- or long-term FVIII expression via PB induced this cellular pathology. ER stress markers, including binding immunoglobulin protein (BiP), the UPR-regulated CCAAT/enhancer-binding protein homologous protein (CHOP), and ER degradation-enhancing α-mannosidase-like protein (EDEM), are elevated after initiation of the unfolded protein response (UPR).19–21 In comparing the abundance of these transcripts at 1 day and 16–24 weeks after gene transfer, we found no evidence of stress in animals receiving PB-coFVIII-BDD and no significant difference between experimental groups and sham controls at either time point (Figure 4).

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