Research ArticleGene Therapy

Liver-directed lentiviral gene therapy in a dog model of hemophilia B

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Science Translational Medicine  04 Mar 2015:
Vol. 7, Issue 277, pp. 277ra28
DOI: 10.1126/scitranslmed.aaa1405
  • Fig. 1. Intraportal administration of lentiviral vectors to dogs with hemophilia B.

    (A) Schematic representation of the third-generation SIN lentiviral vectors (proviral form) used in this work. U3 del, deletion of the promoter/enhancer of the HIV LTR (43); SD, splicing donor site; SA, splicing acceptor site; ψ, packaging signal; wpre*, mutated woodchuck hepatitis virus posttranscriptional regulatory element (44); 142T, miR-142 target sequence made of four tandem copies of a sequence perfectly complementary to miR-142. The hepatocyte-specific ET promoter was composed of synthetic hepatocyte-specific enhancers and the transthyretin promoter (45). cFIX, co-cFIX, and co-cFIXR338L were used as transgenes (14). (B to G) Serum concentrations of alanine aminotransferase (ALT) (B) and aspartate aminotransferase (AST) (C), platelet counts (D), and serum concentrations of tumor necrosis factor–α (TNF-α) (E), interleukin-6 (IL-6) (F), and IL-8 (G) were measured in blood samples collected at the indicated time points after lentiviral vector administration to dogs M57 (gray line), O21 (green line), and O59 (blue line). Baseline values are shown as “time 0.” (B to D) The normal range is shown (dashed lines). (E to G) The means ± SD (gray area) and ranges (dashed lines) of the serum concentrations of each cytokine measured in samples collected from 11 control untreated dogs are shown. Note that the lowest range for TNF-α and IL-6 is 0. Dog O59 was administered corticosteroids and antihistamine drugs before lentiviral vector infusion to reduce inflammation.

  • Fig. 2. Lentiviral vector–mediated gene therapy targeted to the liver provides stable improvement in clotting time in dogs with hemophilia B.

    (A to C) WBCT (A) measured in blood samples and cFIX activity (B) and cFIX antigen (C) measured by aPTT (B) or enzyme-linked immunosorbent assay (ELISA) (C) in plasma samples collected from dogs M57 (gray line), O21 (green line), and O59 (blue line) at the indicated times after lentiviral vector administration. The colored vertical lines indicate 27 days after the last normal plasma transfusion of the dogs at which time exogenous canine factor IX had been washed out. (D) Frequency of spontaneous bleedings (bleeding events per month of observation) in treated dogs after gene therapy. For M57, the frequency of spontaneous bleeding before gene therapy is shown. The mean ± SD bleeding frequency of 10 untreated dogs with hemophilia B (hemoB) in the colony is shown (black bar) (46). P < 0.0001 (two-sample test for equality of proportions; see also table S6).

  • Fig. 3. No evidence of genotoxicity after lentiviral vector integration into the liver of mice.

    (A) Factor IX (FIX) antigen measured by ELISA in the plasma collected from mice early (<3 months) or late (6 to 12 months) after lentiviral vector administration. P = 0.391, Student’s t test. (B) Vector copy number (VCN) in liver DNA collected from mice euthanized early or late after lentiviral vector administration. P = 0.806, Student’s t test. (A and B) Data are means ± SEM. (C) Venn diagram representing CIS identified in liver DNA of mice euthanized early or late after lentiviral vector administration. The overlap is calculated considering the gene associated with each CIS; the number of CIS that passed the Grubbs’ test is shown along with the gene name. The number of samples analyzed and integration sites retrieved are indicated for the two data sets.

  • Fig. 4. SIN.ET lentiviral vectors do not induce HCC in tumor-prone mice.

    (A) Experimental outline of the in vivo biosafety study in mice. Left: Schematic representations of the lentiviral vectors used. Either SIN.ET (gene therapy lentiviral vector with SIN LTRs and an internal ET promoter) or ET.LTR (oncogenic lentiviral vector with transcriptionally active LTRs containing the ET promoter) was administered at matched doses to newborn Cdkn2a−/−Ifnar1−/− tumor-prone mice or wild-type mice resulting in four experimental groups. Wild-type mice then were given a CCl4-based tumor-promoting regimen. Mice were euthanized at the indicated time points or earlier if sick. Necropsy was performed and samples were collected for DNA extraction (for determination of vector copy number and the retrieval of integration sites) and for histopathological analysis. (B and C) Shown is the incidence of HCC in Cdkn2a−/−Ifnar1−/− mice (B) or wild-type mice (C) transduced with the two different lentiviral vectors (SIN.ET or ET.LTR) or untransduced (UNT). Untransduced mice include historical controls (n = 20 Cdkn2a−/−Ifnar1−/− and n = 9 wild-type mice) (27). P values were calculated by two-tailed Fisher’s exact test. Numbers on the histograms indicate the number of mice that developed HCC. (D and E) Vector copy number in liver DNA from Cdkn2a−/−Ifnar1−/− mice (D) or wild-type mice (E) collected 2 weeks after lentiviral vector administration (early) or at necropsy (late). Data are means ± SEM. P values were calculated by one-way analysis of variance (ANOVA) and Bonferroni’s multiple correction test. All vector copy numbers were measured in nontumoral liver tissue except for ET.LTR-induced HCCs.

  • Fig. 5. Integration site analysis does not reveal genotoxicity of SIN.ET lentiviral vectors in tumor-prone mice.

    (A) Venn diagram representing CIS identified in liver DNA of SIN.ET-transduced and ET.LTR-transduced mice. The overlap is calculated considering the gene associated with each CIS. The number of CIS that passed the Grubbs’ test is shown with the gene name (red). The number of samples analyzed and the total number of integration sites are indicated for the two data sets. (B and C) Schematic drawing of two representative CIS of ET.LTR (B) and SIN.ET (C) lentiviral vectors. Each colored bar represents an integration site (red, from ET.LTR-induced HCCs; orange, from nontumoral liver of mice transduced with ET.LTR; black, from liver of mice transduced with SIN.ET). Colored arrows indicate the orientation of the integration site. The gene within the region is represented below, with black boxes indicating exons and arrows indicating transcription orientation. The span of the outlined genomic region is indicated on top. (D) CIS power calculated as the number of different integration sites targeting each CIS. (E) CIS representation calculated as percent of sequencing reads from all integration sites comprised within a CIS over the total number of reads within an experimental data set. (D and E) Data are means ± SEM. P values were calculated by one-way ANOVA and Bonferroni’s multiple correction test. For all the analyses, integration sites from the two mouse models were merged.

  • Table 1. Gene therapy dose response in treated dogs with hemophilia B.

    The table shows the age and weight at treatment of three dogs with hemophilia B (M57, O21, and O59), the infused dose of lentiviral vector in TU and physical particles (p24) per weight, the follow-up (FU) time in days, the whole blood clotting time (WBCT) in minutes, and the cFIX activity [determined by activated partial thromboplastin time (aPTT)]. Also shown are the cFIX antigen (determined by ELISA), the type of transgene contained in the infused lentiviral vector: wild-type (WT) cFIX, co-cFIX, or co-cFIXR338L (see also Fig. 1). When possible, results are presented as mean values ± SEM over time (with ranges in parentheses). The values of WBCT and cFIX activity and antigen are considered valid only if measured 27 days after the last canine plasma transfusion (to ensure washout of exogenous cFIX).

    M57 (Hemil)O21 (Valentine)O59 (Enzo)
    Age at treatment (months)82121
    Weight at treatment (kg)202218
    TransgeneWT cFIXco-cFIXco-cFIXR338L
    TU/kg5.7x1082.3x1091.1x109
    μg p24/kg4447174
    FU (days)1831900637
    WBCT (min)20.31 ± 0.91 (14.5–32)17.36 ± 0.66 (13.5–22.5)15.73 ± 0.5 (11–19.5)
    cFIX activity (% normal)0.08 ± 0.01 (0.01–0.25)1.05 ± 0.12 (0.3–1.7)1.18 ± 0.08 (0.7–1.9)
    cFIX antigen (% normal)0.05 ± 0.004 (0.01–0.09)0.6 ± 0.06 (0.2–0.85)0.16 ± 0.005 (0.14–0.2)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/277/277ra28/DC1

    Materials and Methods

    Fig. S1. Lentiviral vectors efficiently transduce and regulate transgene expression in canine cells.

    Fig. S2. Hematocrit, plasma fibrinogen, TAT, D-dimer, and clearance of lentiviral vector particles from blood after portal vein infusion in dogs with hemophilia B.

    Fig. S3. Histology of liver biopsies from treated dogs with hemophilia B.

    Fig. S4. Lentiviral vector content in liver biopsies and blood and sperm samples from treated dogs with hemophilia B.

    Fig. S5. Factor IX expression and liver histology of tumor-prone mice or mice treated with a tumor promoter transduced with SIN.ET or ET.LTR lentiviral vectors.

    Fig. S6. Comparison of the ET.LTR and SIN.ET CIS under different experimental conditions.

    Table S1. Large-scale batches of lentiviral vectors.

    Table S2. Blood cell counts and blood chemistry in M57.

    Table S3. Blood cell counts and blood chemistry in O21.

    Table S4. Blood cell counts and blood chemistry in O59.

    Table S5. Lentiviral vector particles in swabs from dogs O21 and O59.

    Table S6. Bleeding frequency in treated and untreated dogs with hemophilia B.

    Table S7. Thromboelastography in treated dogs with hemophilia B.

    Table S8. Inhibitor screen in treated dogs with hemophilia B.

    Table S9. Mice with hemophilia B transduced with SIN.ET as adults.

    Table S10. Integration sites retrieved from mice with hemophilia B transduced with SIN.ET as adults.

    Table S11. CIS identified in the data set of integration sites retrieved from mice with hemophilia B transduced as adults.

    Table S12. Cdkn2a−/−Ifnar1−/− and wild-type mice transduced with SIN.ET or ET.LTR as neonates.

    Table S13. Integration sites retrieved from tumor-prone mice transduced as neonates.

    Table S14. CIS identified in the data set of integration sites retrieved from tumor-prone mice transduced as neonates.

    References (4751)

  • Supplementary Material for:

    Liver-directed lentiviral gene therapy in a dog model of hemophilia B

    Alessio Cantore, Marco Ranzani, Cynthia C. Bartholomae, Monica Volpin, Patrizia Della Valle, Francesca Sanvito, Lucia Sergi Sergi, Pierangela Gallina, Fabrizio Benedicenti, Dwight Bellinger, Robin Raymer, Elizabeth Merricks, Francesca Bellintani, Samia Martin, Claudio Doglioni, Armando D'Angelo, Thierry VandenDriessche, Marinee K. Chuah, Manfred Schmidt, Timothy Nichols, Eugenio Montini, Luigi Naldini*

    *Corresponding author. E-mail: naldini.luigi@hsr.it

    Published 4 March 2015, Sci. Transl. Med. 7, 277ra28 (2015)
    DOI: 10.1126/scitranslmed.aaa1405

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Lentiviral vectors efficiently transduce and regulate transgene expression in canine cells.
    • Fig. S2. Hematocrit, plasma fibrinogen, TAT, D-dimer, and clearance of lentiviral vector particles from blood after portal vein infusion in dogs with hemophilia B.
    • Fig. S3. Histology of liver biopsies from treated dogs with hemophilia B.
    • Fig. S4. Lentiviral vector content in liver biopsies and blood and sperm samples from treated dogs with hemophilia B.
    • Fig. S5. Factor IX expression and liver histology of tumor-prone mice or mice treated with a tumor promoter transduced with SIN.ET or ET.LTR lentiviral
      vectors.
    • Fig. S6. Comparison of the ET.LTR and SIN.ET CIS under different experimental conditions.
    • Table S1. Large-scale batches of lentiviral vectors.
    • Table S2. Blood cell counts and blood chemistry in M57.
    • Table S3. Blood cell counts and blood chemistry in O21.
    • Table S4. Blood cell counts and blood chemistry in O59.
    • Table S5. Lentiviral vector particles in swabs from dogs O21 and O59.
    • Table S6. Bleeding frequency in treated and untreated dogs with hemophilia B.
    • Table S7. Thromboelastography in treated dogs with hemophilia B.
    • Table S8. Inhibitor screen in treated dogs with hemophilia B.
    • Legends for tables S9 to S14
    • References (4751)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S9 (Microsoft Excel format). Mice with hemophilia B transduced with SIN.ET as adults.
    • Table S10 (Microsoft Excel format). Integration sites retrieved from mice with hemophilia B transduced with SIN.ET as adults.
    • Table S11 (Microsoft Excel format). CIS identified in the data set of integration sites retrieved from mice with hemophilia B transduced as adults.
    • Table S12 (Microsoft Excel format). Cdkn2a−/−Ifnar1−/− and wild-type mice transduced with SIN.ET or ET.LTR as neonates.
    • Table S13 (Microsoft Excel format). Integration sites retrieved from tumor-prone mice transduced as neonates.
    • Table S14 (Microsoft Excel format). CIS identified in the data set of integration sites retrieved from tumor-prone mice transduced as neonates.

    [Download Tables S9 to S14]

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