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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

Hyperactive piggyBac transposase-mediated FVIII expression persists in vivo. Twenty-five microgram PB-coFVIII-BDD transposon was given in a 1:1 ratio with either iPB7 (box, n = 19) or inactive transposase (inverted triangle, PBD268L, n = 8) to FVIII  mice. DNA was prepared in 2 ml Lactated Ringer’s solution (LR) and delivered hydrodynamically to 6- to 8-week-old FVIII  mice. Results for LR-treated FVIII  mice (shaded circle, n = 12) and wild-type mice (triangle, n = 15) are also indicated. (a) FVIII activity and (b) antigen were measured using the Coamatic activity assay or enzyme-linked immunosorbent assay (ELISA) respectively. Points indicate means ± SE. *P < 0.001 and **P = 0.002, both measured via a one-way analysis of variance. (c) Mice treated with PB-coFVIII-BDD + iPB7 were assessed for inhibitor development. Three mice revealed low-titer inhibitors. All other mice had Bethesda titers of <0.5. (Bethesda titers >0.5 are clinically relevant.) This graph represents FVIII activity without these three mice and reveals FVIII levels indistinguishable from wild-type mice. (d) Twenty-five microgram PB-coFVIII-BDD transposon (box, n = 8) or PB-FVIII-BDD (open circle, n = 9) was given in a 1:1 ratio with iPB7 to FVIII  mice as described above. Results for LR-treated FVIII  mice (filled circle, n = 7) are also indicated. FVIII activity was measured using the Coamatic activity assay. Points indicate means ± SE. ***P < 0.001, measured via a one-way analysis of variance.
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fig3: Hyperactive piggyBac transposase-mediated FVIII expression persists in vivo. Twenty-five microgram PB-coFVIII-BDD transposon was given in a 1:1 ratio with either iPB7 (box, n = 19) or inactive transposase (inverted triangle, PBD268L, n = 8) to FVIII mice. DNA was prepared in 2 ml Lactated Ringer’s solution (LR) and delivered hydrodynamically to 6- to 8-week-old FVIII mice. Results for LR-treated FVIII mice (shaded circle, n = 12) and wild-type mice (triangle, n = 15) are also indicated. (a) FVIII activity and (b) antigen were measured using the Coamatic activity assay or enzyme-linked immunosorbent assay (ELISA) respectively. Points indicate means ± SE. *P < 0.001 and **P = 0.002, both measured via a one-way analysis of variance. (c) Mice treated with PB-coFVIII-BDD + iPB7 were assessed for inhibitor development. Three mice revealed low-titer inhibitors. All other mice had Bethesda titers of <0.5. (Bethesda titers >0.5 are clinically relevant.) This graph represents FVIII activity without these three mice and reveals FVIII levels indistinguishable from wild-type mice. (d) Twenty-five microgram PB-coFVIII-BDD transposon (box, n = 8) or PB-FVIII-BDD (open circle, n = 9) was given in a 1:1 ratio with iPB7 to FVIII mice as described above. Results for LR-treated FVIII mice (filled circle, n = 7) are also indicated. FVIII activity was measured using the Coamatic activity assay. Points indicate means ± SE. ***P < 0.001, measured via a one-way analysis of variance.

Mentions: We next evaluated the persistence of FVIII expression after transposon delivery. Three days after delivery, animals receiving PB-coFVIII-BDD + iPB7 or PB-coFVIII-BDD + inactive transposase demonstrated FVIII levels greater than 50%. By 2 weeks after injection, mice receiving PB-coFVIII-BDD + inactive transposase had FVIII activity of 0% similar to mice that received LR (P = 0.5). Mice receiving LR alone had undetectable levels of FVIII (0%) throughout the study. Animals receiving PB-coFVIII-BDD + iPB7 had significantly higher FVIII activity than mice receiving LR alone (P < 0.001) or animals receiving inactive transposase (P < 0.05, Figure 3a). FVIII levels in the transposon + iPB7 treated animals were similar to wild type (P = 0.9, Figure 3a) and more importantly, this activity was sustained for the study duration. At 24 weeks after injection, PB-coFVIII-BDD + iPB7 treated mice averaged FVIII activity of 57% compared to pooled human plasma.


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)

Hyperactive piggyBac transposase-mediated FVIII expression persists in vivo. Twenty-five microgram PB-coFVIII-BDD transposon was given in a 1:1 ratio with either iPB7 (box, n = 19) or inactive transposase (inverted triangle, PBD268L, n = 8) to FVIII  mice. DNA was prepared in 2 ml Lactated Ringer’s solution (LR) and delivered hydrodynamically to 6- to 8-week-old FVIII  mice. Results for LR-treated FVIII  mice (shaded circle, n = 12) and wild-type mice (triangle, n = 15) are also indicated. (a) FVIII activity and (b) antigen were measured using the Coamatic activity assay or enzyme-linked immunosorbent assay (ELISA) respectively. Points indicate means ± SE. *P < 0.001 and **P = 0.002, both measured via a one-way analysis of variance. (c) Mice treated with PB-coFVIII-BDD + iPB7 were assessed for inhibitor development. Three mice revealed low-titer inhibitors. All other mice had Bethesda titers of <0.5. (Bethesda titers >0.5 are clinically relevant.) This graph represents FVIII activity without these three mice and reveals FVIII levels indistinguishable from wild-type mice. (d) Twenty-five microgram PB-coFVIII-BDD transposon (box, n = 8) or PB-FVIII-BDD (open circle, n = 9) was given in a 1:1 ratio with iPB7 to FVIII  mice as described above. Results for LR-treated FVIII  mice (filled circle, n = 7) are also indicated. FVIII activity was measured using the Coamatic activity assay. Points indicate means ± SE. ***P < 0.001, measured via a one-way analysis of variance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Hyperactive piggyBac transposase-mediated FVIII expression persists in vivo. Twenty-five microgram PB-coFVIII-BDD transposon was given in a 1:1 ratio with either iPB7 (box, n = 19) or inactive transposase (inverted triangle, PBD268L, n = 8) to FVIII mice. DNA was prepared in 2 ml Lactated Ringer’s solution (LR) and delivered hydrodynamically to 6- to 8-week-old FVIII mice. Results for LR-treated FVIII mice (shaded circle, n = 12) and wild-type mice (triangle, n = 15) are also indicated. (a) FVIII activity and (b) antigen were measured using the Coamatic activity assay or enzyme-linked immunosorbent assay (ELISA) respectively. Points indicate means ± SE. *P < 0.001 and **P = 0.002, both measured via a one-way analysis of variance. (c) Mice treated with PB-coFVIII-BDD + iPB7 were assessed for inhibitor development. Three mice revealed low-titer inhibitors. All other mice had Bethesda titers of <0.5. (Bethesda titers >0.5 are clinically relevant.) This graph represents FVIII activity without these three mice and reveals FVIII levels indistinguishable from wild-type mice. (d) Twenty-five microgram PB-coFVIII-BDD transposon (box, n = 8) or PB-FVIII-BDD (open circle, n = 9) was given in a 1:1 ratio with iPB7 to FVIII mice as described above. Results for LR-treated FVIII mice (filled circle, n = 7) are also indicated. FVIII activity was measured using the Coamatic activity assay. Points indicate means ± SE. ***P < 0.001, measured via a one-way analysis of variance.
Mentions: We next evaluated the persistence of FVIII expression after transposon delivery. Three days after delivery, animals receiving PB-coFVIII-BDD + iPB7 or PB-coFVIII-BDD + inactive transposase demonstrated FVIII levels greater than 50%. By 2 weeks after injection, mice receiving PB-coFVIII-BDD + inactive transposase had FVIII activity of 0% similar to mice that received LR (P = 0.5). Mice receiving LR alone had undetectable levels of FVIII (0%) throughout the study. Animals receiving PB-coFVIII-BDD + iPB7 had significantly higher FVIII activity than mice receiving LR alone (P < 0.001) or animals receiving inactive transposase (P < 0.05, Figure 3a). FVIII levels in the transposon + iPB7 treated animals were similar to wild type (P = 0.9, Figure 3a) and more importantly, this activity was sustained for the study duration. At 24 weeks after injection, PB-coFVIII-BDD + iPB7 treated mice averaged FVIII activity of 57% compared to pooled human plasma.

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