Figure 1.
piggyBac vectors to express Factor VIII cDNAs.
(A) Schematic diagram of piggyBac vectors expressing EGFP, B-domain–deleted human FVIII, and full-length human FVIII under the control of the human EF1α promoter. The PBaseII vector expresses piggyBac transposase under the control of the CAG promoter. IRES: internal ribosomal entry site. (B) Copy number of genomic piggyBac vectors. Indicated piggyBac vectors were transfected into 293T cells and selected with puromycin resistance. Approximately three months after transduction, genomic DNAs were extracted, and piggyBac vector copy numbers were assessed by real-time PCR using the piggyBac 5′ TR primers. The data are normalized to a haploid genome calculated from the copy number of NANOG gene. *: P<0.05 by two-sided Student's t test (n = 3). N.S.: Not significant. (C) The sizes of inserted FVIII cDNA (2,219 bp for BDD and 4,901 bp for full-length FVIII) were confirmed by genomic PCR using the hF8insertC primers flanking the B-domain (indicated as small arrows in Figure 1A).
Figure 2.
Full-length FVIII can be expressed at levels as high as BDD FVIII.
(A, B) Total mRNAs were extracted from 293T cells (A) or human iPS cells (B), and the level of expression was assessed by quantitative RT-PCR. Expression values were normalized to the level of GAPDH mRNA. (C, D) Secretion of functional FVIII measured by aPTT assay. Culture supernatants of transfected cells were harvested 3 days after medium change and subjected to aPTT assay to measure the coagulation activity of secreted FVIII protein in 293T cells (C) or human iPS cells (D). Recombinant FVIII product was used to generate a standard curve. Normal FVIII activity (100%) represents 1 U/ml ( = 1000 mU/ml). *: P<0.05 by two-sided Student's t test (n = 3).
Figure 3.
Long-term and stable expression of FVIII in hemophilia A mice.
(A, B) piggyBac vector (25 µg of DNA) expressing either B-domain–deleted (A) or full-length FVIII (B) was introduced into hemophilia A mice (n = 5 each) by hydrodynamic injection without cyclophosphamide treatments. Amount of PBaseII piggyBac transposase (0 µg, 6.25 µg, or 25 µg of DNA) is indicated by symbols. Levels of anti-human FVIII inhibitors in mouse plasma were measured by Bethesda assay. (C, D) piggyBac vector (25 µg of DNA) expressing either B-domain-deleted (C) or full-length FVIII (D) was introduced into hemophilia A mice (n = 7 each) by hydrodynamic injection with cyclophosphamide treatments. Amount of PBaseII piggyBac transposase (0 µg, 6.25 µg or 25 µg of DNA) is indicated by symbols. Levels of FVIII activity in mouse plasma were measured by chromogenic assay. (E, F) Multiple injections boosted the level of FVIII in hemophilia A mice. PB-EF1α-hFVIII(Full)-iP (25 µg) and PBaseII (6.25 µg) vectors were hydrodynamically injected into hemophilia A mice (n = 7 each) three times at intervals of 24 hours or 4 weeks. (E) Level of FVIII activity in mouse plasma was measured by chromogenic assay. (F) Level of anti-human FVIII inhibitor was measured by Bethesda assay.
Figure 4.
Phenotypic correction of hemophilia A mice by injection of piggyBac vectors.
(A) Bleeding time of both hemophilia A mice (n = 6) and hemophilia A mice treated with piggyBac vector expressing full-length Factor VIII (n = 5) assessed by tail-clip assay. (B) Immunohistochemical analysis of liver tissue from non-treated hemophilia A mice and hemophilia A mice treated with piggyBac vector expressing full-length FVIII. Scale bar represents 50 µm (x400: original magnification). (C) Total RNA were extracted from the mouse liver treated with piggyBac vectors with 4 weeks interval, and quantified the level of transgene mRNA by qRT-PCR using human FVIII light-chain primers. Expression values were normalized to the level of GAPDH mRNA.