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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

piggyBac gene transfer improved survival in vivo. Twenty-five microgram transposon with the liver-specific FVIII expression cassette in a 1:1 ratio with hyperactive or inactive transposase or Lactated Ringer’s (LR) alone were delivered hydrodynamically to 6- to 8-week-old FVIII  mice and survival monitored. Mice injected with iPB7 (n = 19) had significantly improved survival compared to those injected with Lactated Ringer’s alone or inactive transposase (*P < 0.02, log-rank (Mantel-Cox) test). Uninjected animals (n = 21) demonstrated a similar survival to those injected with LR (n = 12) or inactive transposase (n = 8).
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fig2: piggyBac gene transfer improved survival in vivo. Twenty-five microgram transposon with the liver-specific FVIII expression cassette in a 1:1 ratio with hyperactive or inactive transposase or Lactated Ringer’s (LR) alone were delivered hydrodynamically to 6- to 8-week-old FVIII mice and survival monitored. Mice injected with iPB7 (n = 19) had significantly improved survival compared to those injected with Lactated Ringer’s alone or inactive transposase (*P < 0.02, log-rank (Mantel-Cox) test). Uninjected animals (n = 21) demonstrated a similar survival to those injected with LR (n = 12) or inactive transposase (n = 8).

Mentions: Previous descriptions of the hemophilia A mouse models indicated a normal lifespan and lethal bleeding with procedures such as tail clip.17 However, we observed a shortened lifespan in hemophilia A mice receiving LR or inactive transposase after hydrodynamic tail-vein injection, an intervention known to cause liver trauma.18 All mice receiving PB-coFVIII-BDD + iPB7 survived for the duration of the study (either 16 or 24 weeks). In contrast, only 8 of 12 mice receiving LR (67%) and 5 of 8 mice receiving inactive transposase (62.5%) survived for the duration of the study (Figure 2, P = 0.02). To determine if the hydrodynamic tail-vein injection altered the survival of animals injected with LR or inactive transposase, a group of untreated animals were included. These animals demonstrated an overall survival of 52% which was not statistically different from the groups receiving LR or inactive transposase. These data suggest that the hemophilia A mouse may have a shortened lifespan and FVIII expression improved outcomes after gene delivery in these mice.


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)

piggyBac gene transfer improved survival in vivo. Twenty-five microgram transposon with the liver-specific FVIII expression cassette in a 1:1 ratio with hyperactive or inactive transposase or Lactated Ringer’s (LR) alone were delivered hydrodynamically to 6- to 8-week-old FVIII  mice and survival monitored. Mice injected with iPB7 (n = 19) had significantly improved survival compared to those injected with Lactated Ringer’s alone or inactive transposase (*P < 0.02, log-rank (Mantel-Cox) test). Uninjected animals (n = 21) demonstrated a similar survival to those injected with LR (n = 12) or inactive transposase (n = 8).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: piggyBac gene transfer improved survival in vivo. Twenty-five microgram transposon with the liver-specific FVIII expression cassette in a 1:1 ratio with hyperactive or inactive transposase or Lactated Ringer’s (LR) alone were delivered hydrodynamically to 6- to 8-week-old FVIII mice and survival monitored. Mice injected with iPB7 (n = 19) had significantly improved survival compared to those injected with Lactated Ringer’s alone or inactive transposase (*P < 0.02, log-rank (Mantel-Cox) test). Uninjected animals (n = 21) demonstrated a similar survival to those injected with LR (n = 12) or inactive transposase (n = 8).
Mentions: Previous descriptions of the hemophilia A mouse models indicated a normal lifespan and lethal bleeding with procedures such as tail clip.17 However, we observed a shortened lifespan in hemophilia A mice receiving LR or inactive transposase after hydrodynamic tail-vein injection, an intervention known to cause liver trauma.18 All mice receiving PB-coFVIII-BDD + iPB7 survived for the duration of the study (either 16 or 24 weeks). In contrast, only 8 of 12 mice receiving LR (67%) and 5 of 8 mice receiving inactive transposase (62.5%) survived for the duration of the study (Figure 2, P = 0.02). To determine if the hydrodynamic tail-vein injection altered the survival of animals injected with LR or inactive transposase, a group of untreated animals were included. These animals demonstrated an overall survival of 52% which was not statistically different from the groups receiving LR or inactive transposase. These data suggest that the hemophilia A mouse may have a shortened lifespan and FVIII expression improved outcomes after gene delivery in these mice.

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