1. Long-Term and Therapeutic-Level Hepatic Gene Expression of Human Factor IX after Naked Plasmid Transfer in Vivo 
Carol H. Miao, Arthur R. Thompson, Keith Loeb, Xin Ye 
Molecular Therapy 
Volume 3, Issue 6, Pages (June 2001)
DOI: /mthe
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
(A) The structure of constructs containing different hFIX expression cassettes. The plasmid pBS-HCRHP-FIXIA is abbreviated from pBS-ApoEHCR-hAATp-hFIX+IntA-bpA (12), and pBS-HP-FIXA is abbreviated from pBS-hAATp-hFIX-bpA. HCR, hepatic locus control region from the ApoE gene locus (771 bp); hAAT(P), human α1-anti-trypsin promoter (408 bp); hFIX, human factor IX cDNA (1.4 kb); Intron A, truncated human factor IX first intron (1.4 kb); bpA, bovine growth hormone polyadenylation signal (265 bp). (B–D) Expression Levels of hFIX over time after naked DNA transfer into mouse livers by two different plasmids shown in (A). Mice were rapidly (5–8 s) infused with 20 μg of plasmid in 2 ml saline into the tail vein. Groups of mice (n = 3/group) treated with each plasmid were sacrificed at four different time points postinjection, 1 day (data not shown), 3 days (data not shown), (B) 30 days, and (C) 240 days. (D) Human FIX levels were followed for the rest of the treated mice for the duration of the experiments (550 days). Filled symbols, pBS-HCRHP-FIXIA; open symbols, pBS-HP-FIXA.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
DNA and RNA analyses. (A) Southern analyses. Total cellular DNA was isolated from different organs of four groups of mice (n = 3, one representative animal shown from each group) that had received plasmid DNAs, pBS-HP-FIXA or pBS-HCRHP-FIXIA, as described in Fig. 1. Each group was killed at a different time point (day 1, 3, 30, or 240). DNA was then digested with PstI and analyzed by Southern blot with a radiolabeled human factor IX probe. Lane 1, liver DNA from a naïve mouse; lane 2, 100 pg of pBS-HCRHP-FIXIA; lanes 3–9, DNA from different organs as marked on the graph from pBS-HCRHP-FIXIA-treated mouse; lane 10, 100 pg of pBS-HP-FIXA; lanes 11–17, DNA from different organs as marked from pBS-HCRHP-FIXIA-treated mouse. (B) RT-PCR analyses. The mRNA was isolated from different tissues of four groups of mice (n = 3 mice/group, one representative animal shown) that received plasmid pBS-HP-FIXA or pBS-HCRHP-FIXIA. The isolated transcripts were subjected to RT-PCR analysis using primers complementary to sequences in exons 2–8 of the hFIX cDNA spanning 810 bp (top) and primers complementary to β-actin mRNA (bottom) to confirm that the mRNA was intact in all samples. The PCR products were electrophoresed on a 1% ethidium bromide gel. Lane 1, positive control, DNA-based PCR using the plasmid as a template; lane 2, actin control; lane 3, liver RNA treated in an identical manner without reverse transcriptase; lane 4, liver RNA from a naïve mouse; lanes 5–11, mRNA from different organs as marked from pBS-HCRHP-FIXIA-treated mouse; lanes 12–18, mRNA from different organs as marked from pBS-HP-FIXA-treated mouse.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
DNA and mRNA in mouse livers 3 days after slower injection (30 s) of plasmid DNA pBS-HCRHP-FIXIA. (A) Southern analyses. Total cellular DNA was isolated from different tissues of three mice (one representative animal shown) that had received plasmid pBS-HCRHP-FIXIA. The plasmid was injected in 2 ml saline into the tail vein of mouse in 30 s. The DNA was analyzed the same way as described for Fig. 2A. Lane 1, 100 pg of pBS-HCRHP-FIXIA; lane 2, liver DNA from a naive mouse; lanes 3–9, DNA from different organs as marked. (B) RT-PCR analyses. RNA isolated from different tissues of three mice (one representative animal shown) that had received plasmid pBS-HCRHP-FIXIA with slower injection was subjected to RT-PCR analyses the same say as described for Fig. 2B. Lane 1, positive control, DNA-based PCR using the plasmid as a template; lane 2, actin control; lane 3, liver RNA treated in an identical manner without reverse transcriptase; lane 4, liver RNA from a naive mouse; lanes 5–11, mRNA from different organs as marked from pBS-HCRHP-FIXIA-treated mouse.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Southern analysis of the liver samples. Total cellular DNA was isolated from the livers of four groups of mice (n = 3, one representative animal shown from each group) treated as described for Fig. 1. DNA from naive mice with or without added pBS-HCRHP-FIXIA or pBS-HP-FIXA was used as positive and negative controls. DNA samples (10 μg) were digested with ClaI (A), digested with PstI (B), or undigested (C) and analyzed by Southern blot. Hybridization was with a 32P-radiolabeled 810-nt F9 fragment extending from exon II to exon VIII of the hFIX cDNA. Lanes 1 and 2, 20 and 100 pg of pBS-HCRHP-FIXIA; lane 3, liver DNA from naive mouse; lanes 4–7, liver DNA from mice sacrificed at different time points after plasmid pBS-HCRHP-FIXIA injections as marked; lanes 8 and 9, 20 and 100 pg of pBS-HP-FIXA; lane 10, liver DNA from naive mouse; lanes 11–14, liver DNA from mice sacrificed at different time points after plasmid pBS-HP-FIXA injections as marked. (D) Total liver DNA was separated into chromosomal and extrachromosomal fractions using a modified Hirt procedure (15). These fractions were digested with PstI and analyzed by Southern blot the same way as described above. Lanes 1 and 2, 20 and 100 pg of pBS-HCRHP-FIXIA; lane 3, liver DNA from naive mouse; lanes 4 and 5, liver DNA from naïve mouse with added 50 pg pBS-HCRHP-FIXIA; lane 4, Hirt fraction; lane 5, chromosomal fraction; lanes 6 and 7, samples from groups of mice (n = 3/group, one representative animal shown) treated with pBS-HCRHFIXIA; lane 6, Hirt fraction; lane 7, chromosomal fraction.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
HFIX expression levels in mice which underwent partial hepatectomy. Two mice, K3 and K5, had persistent hFIX gene expression levels (0.3 and 0.8 μg/ml) 250 days after rapid plasmid infusion with pBS-B2. These two mice were then subjected to partial hepatectomy to remove two-thirds of their livers. HFIX levels were then followed after surgery over time. Both mice were sacrificed 1 month after partial hepatectomy, and their livers were found to have regenerated back to the normal size.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Histology of the liver sections. Livers isolated from four groups of mice that had received 20 μg of plasmid pBS-HCRHP-FIXIA or saline-only control and killed at different time points after injection were sectioned and examined by routine hematoxylin and eosin staining. (A, B, C, D) From livers of plasmid-treated mice, (A) day 1, (B) day 3, (C) day 10, (D) day 180. (E, F, G, H) From livers of saline-treated mice, (E) day 1, (F) day 3, (G) day 10, (H) day 180. (A, B, C, E, F, and G) are with high-power magnification to show the lesion sites, whereas (D and H) are with low-power magnification. Arrows point to some of the focal regions of hemorrhage.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions

8. FIG. 7
Antibody formation against hFIX over time in mice treated with plasmid pBS-HCRHP-FIXIA (n = 10). Antibody titer in μg IgG/ml was determined by antigen-specific ELISA. Each line represents an individual animal.
Molecular Therapy 2001 3, DOI: ( /mthe )
Copyright © 2001 American Society for Gene Therapy Terms and Conditions