1. Volume 10, Issue 1, Pages 117-126 (July 2004)
Naked DNA Transfer of Factor VIII Induced Transgene-Specific, Species-Independent Immune Response in Hemophilia A Mice 
Peiqing Ye, Arthur R. Thompson, Rita Sarkar, Zhenping Shen, David P. Lillicrap, Randal J. Kaufman, Hans D. Ochs, David J. Rawlings, Carol H. Miao 
Molecular Therapy 
Volume 10, Issue 1, Pages (July 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Liver-specific FVIII constructs carrying cDNAs from human, canine, and murine origins. B-domain-deleted human, canine, and murine cDNAs were each inserted into a liver-specific vector, pBS-HCRHPI-A, to generate constructs encoding corresponding species-specific FVIII protein, pBS-HCRHPI-FVIIIA, pBS-HCRHPI-cFVIIIA, and pBS-HCRHPI-mFVIIIA. *A B-domain-deleted human FVIII cDNA was inserted into the multiple cloning site of the liver-specific vector. †A B-domain-deleted canine FVIII cDNA was used. §A B-domain-deleted murine FVIII cDNA was used.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Naked plasmid transfer of hFVIII plasmids into hemophilia A mice. (A) Human FVIII levels over time in hemophilia A mice after treatment with pBS-HCRHPI-FVIIIA. Fifty micrograms of the plasmid in 2 ml of saline solution was injected into the tail vein of mice (n = 6) in 5–8 s. Mice were then bled at regular intervals. Circulating hFVIII activities in plasma were evaluated by COATEST and a modified clotting assay. (B) Inhibitor antibody formation against hFVIII over time. (C) Southern analysis of the vector DNA isolated from the treated mouse livers. The arrows indicate different episomal forms of undigested vector DNA. (D) Western analysis of hFVIII protein in the liver at different time points. The upper and lower arrows in the Western blot show the respective 90-kDa heavy chain and 80-kDa light chain of B-domain-deleted hFVIII. (E) Immunofluorescent staining of hepatic hFVIII (original magnification ×40). (a) Untreated hemophilia A mouse liver was used as a negative control. Plasmid-treated mouse livers were harvested (b) 1 day, (c) 60 days, or (d) 120 days postinjection. Transduction efficiency was quantitated for each group (n = 3) and average value is listed at the bottom for each time point.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Naked plasmid transfer of canine FVIII plasmid pBS-HCRHPI-cFVIIIA into hemophilia A mice. (A) Canine FVIII levels over time in hemophilia A mice after plasmid treatment. Fifty micrograms of the plasmid in 2 ml of saline solution was injected into the tail vein of mice (n = 5) in 5–8 s. Circulating cFVIII activities in plasma were evaluated by COATEST and a modified clotting assay. (B) Inhibitory antibody formation against cFVIII over time.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Naked plasmid transfer of murine FVIII plasmid pBS-HCRHPI-mFVIIIA into hemophilia A mice. (A) Murine FVIII levels over time in hemophilia A mice after plasmid treatment. Fifty micrograms of the plasmid in 2 ml of saline solution was injected into the tail vein of mice (n = 5) in 5–8 s. Circulating mFVIII activities in plasma were evaluated by COATEST and a modified clotting assay. (B) Inhibitor antibody formation against mFVIII over time.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Subclasses of anti-hFVIII IgG immunoglobulins in hemophilia A mice after naked plasmid transfer of pBS-HCRHPI-FVIIIA. Titers of subclasses of IgG produced after gene transfer were determined by IgG subclass-specific ELISA. (A) IgG1, (B) IgG2a, (C) IgG2b, (D) IgG3. Each line represents an individual animal.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Naked plasmid transfer of hFVIII plasmids into hemophilia A mice with transient immunosuppression. (A) Human FVIII levels over time in hemophilia A mice after treatment with pBS-HCRHPI-FVIIIA and transient immunosuppression with cyclophosphamide. Fifty micrograms of the plasmid in 2 ml of saline solution was injected into the tail vein of mice (n = 6) in 5–8 s. Cyclophosphamide (50 mg/kg) was administered intraperitoneally at the time of vector injection and 14 and 28 days postinjection. Mice were then bled at regular intervals. Circulating hFVIII activities in plasma were evaluated by COATEST and a modified clotting assay. (B) Inhibitor antibody formation against hFVIII over time.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions