Efficient production of human FVIII in hemophilic mice using lentiviral vectors  Neeltje A Kootstra, Ryusuke Matsumura, Inder M Verma  Molecular Therapy  Volume 7, Issue 5, Pages 623-631 (May 2003) DOI: 10.1016/S1525-0016(03)00073-X Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 In vitro production of human FVIII by LV-transduced cells. (A) Schematic presentation of LV constructs expressing the human B-domain-deleted FVIII cDNA from the CAG promoter, which consists of the chicken β-actin promoter, CMV enhancers, and a large synthetic intron (LV-CAG-FVIIIB). In a second vector the FVIII secretory peptide (aa 1–19) was replaced by the secretory peptide of α-feto protein (aa 1–20) (LV-CAG-AFP-FVIIIB). The LV construct contains the following cis-acting sequences: the packaging signal (Ψ), the Rev-responsive element (RRE), a polypurine tract (cPPT), and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. The arrow depicts the start of transcription. (B) 293T cells were transduced with LV-CAG-FVIIIB and LV-CAG-AFP-FVIIIB at a concentration of 1 × 107 IU per 1 × 105 cells. Forty-eight hours after transduction, culture supernatants and cells were harvested and analyzed for FVIII production by Western blot. Cell lysates and supernatants were run on 3–8% Tris acetate gels and blotted on PVDF membranes. FVIII proteins were identified with a polyclonal antibody against FVIII, a HRP-labeled secondary antibody, and ECL detection reagent. (C) The biological activity of FVIII present in the culture supernatants of the 293T cells harvested 24 h after transduction was analyzed by APTT in seconds. Samples containing high FVIII activity were reanalyzed at a 1:10 dilution and the calculated FVIII concentration has been corrected for this. (D) Primary mouse hepatocytes were transduced with LV-CAG-FVIIIB and LV-CAG-AFP-FVIIIB at a concentration of 1 × 107 IU per 1 × 105 cells. FVIII production was analyzed at days 1 and 5 after transduction with the APTT assay. (E) FVIII production by cells from hematopoietic origin. Lin− and Lin+ cells obtained from human cord blood were transduced with LV-CAG-FVIIIB or LV-CAG-AFP-FVIIIB at a concentration of 1 × 107 IU per 1 × 105 cells. Lin− cells were expanded in medium containing rhIL-3, rhIL-6, and rhSCF, and Lin+ cells were cultured in the presence of PHA and rhIL-2. FVIII activity was detected in the culture supernatants by the APTT assay 24 h after transduction. In all experiments, untransduced cells or cells transduced with a GFP-expressing LV (LV-CAG-GFP) were used as negative controls. Gray bars, FVIII activity analyzed by APTT in seconds. Black bars, FVIII concentration corresponding to the observed FVIII activity calculated using a standard curve and given in mU FVIII/1 × 105 cells/24 h. FL, full-length chain; HC, heavy chain; LC, light chain. Molecular Therapy 2003 7, 623-631DOI: (10.1016/S1525-0016(03)00073-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Low FVIII activity in the plasma of hemophilic mice transduced with LV through systemic administration or BM transplantation due to FVIII inhibitory antibodies. (A) FVIII-expressing LV was delivered in FVIII knockout mice (n = 10) by ip injection at a dose of 1 × 109 IU per mouse. A second group of knockout mice (n = 8) was γ-irradiated (900 cGy) and received 5 × 105 mouse BM cells transduced with 5 × 108 IU of LV-CAG-FVIIIB, through iv injection. FVIII activity in the blood plasma of the mice was analyzed frequently by APTT in seconds over a period of 4 months. Baseline APTT value of FVIII knockout mice is 95.5 ± 7.5 s (n = 28), FVIII knockout plasma supplemented with 50 or 1000 mU concentrated plasma FVIII gives an APTT of 79.1 ± 4.6 and 62.2 ± 4.0 s, respectively (n = 13). (B) The presence of FVIII inhibitors in the blood plasma of transduced mice and controls was analyzed using the Bethesda assay. Plasma samples taken 2 months after transduction were incubated with HPP and after 1 h, the remaining FVIII activity was analyzed by APTT. Plasma samples were diluted until an inhibition of 50% was reached. Results are given as Bethesda units (1 Bethesda unit is neutralization of 50% of the FVIII activity by undiluted plasma). Human FVIII-deficient plasma and plasma obtained from untransduced knockout mice were used as controls in this assay. (C) FACS analysis of the blood mononuclear cells taken from the γ-irradiated (900 cGy) transplantation controls (normal C57BL/6) that received 5 × 105 bone marrow cells from CD45.1 C57BL/6 mice transduced with LV-CAG-GFP (5 × 108 IU), at day 55 after transplantation. White bar, % CD45.1-positive cells in the total mononuclear cell fraction; gray bar, % GFP-expressing cells in the CD45.1-positive cell fraction; black bar, % CD11b-, CD19-, and CD3-positive cells in the CD45.1/GFP-positive cell fraction. Molecular Therapy 2003 7, 623-631DOI: (10.1016/S1525-0016(03)00073-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Efficient LV transduction and FVIII production were observed in the tissues of the mice transduced by systemic delivery or by BM transplantation. (A) Ten weeks after transduction, one animal of each group was sacrificed and the remaining animals were sacrificed at 4 months after transduction. Total DNA was isolated from liver, spleen, blood, and BM and analyzed for the presence of proviral DNA. Proviral LV DNA was detected using a primer pair amplifying a 637-bp fragment of the FVIII cDNA and, as a control for the general efficiency of the PCR, a 220-bp fragment of the β-actin gene was amplified. (B) Tissue samples obtained 10 weeks after transduction were analyzed for the presence of human B-domain-deleted FVIII by Western blot analysis. Ten micrograms of total protein from the tissues was run on 3–8% Tris acetate gels and blotted on PVDF membranes. FVIII proteins were identified with a polyclonal antibody against FVIII, a HRP-labeled secondary antibody, and ECL detection reagent. HC, heavy chain; FL, full-length chain. Molecular Therapy 2003 7, 623-631DOI: (10.1016/S1525-0016(03)00073-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Efficient FVIII production by BM and blood mononuclear cells in ex vivo cultures. (A) Mononuclear cells from BM and blood were isolated from the two mice (one of each group) that were sacrificed 10 weeks after the start of the experiment. Mononuclear cells (1 × 105) were cultured in the presence of rmIL-3, rhIL-6, and rmSCF, and FVIII activity was analyzed by APTT in the culture supernatant during the subsequent days. FVIII activity in the supernatant of BM and blood mononuclear cells was analyzed at 48 and 96 h after isolation, respectively. As a negative control, untransduced BM cells were used. Gray bars, FVIII activity analyzed by APTT in seconds. Black bars, FVIII concentration corresponding to the observed FVIII activity calculated using a standard curve and given in mU FVIII/1 × 105 cells/24 h. (B) Culture supernatants from the blood mononuclear cells were harvested 96 h after the start of the culture and analyzed for the presence of FVIII by Western blot. Samples were run on 3–8% Tris acetate gels and blotted on PVDF membranes. FVIII proteins were identified with a polyclonal antibody against FVIII, a HRP-labeled secondary antibody, and ECL detection reagent. The culture supernatant from untransduced BM cells was used as a negative control. HC, heavy chain; LC, light chain. Molecular Therapy 2003 7, 623-631DOI: (10.1016/S1525-0016(03)00073-X) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions