Research ArticleHEALTH AND MEDICINE

Durable multitransgene expression in vivo using systemic, nonviral DNA delivery

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Science Advances  27 Nov 2019:
Vol. 5, no. 11, eaax0217
DOI: 10.1126/sciadv.aax0217
  • Fig. 1 One intravenous HEDGES hG-CSF cDNA injection produces prolonged hG-CSF protein production and bioactivity in mice.

    (A) Serum hG-CSF levels are shown from groups of mice (three mice per group) treated with liposomes followed by either an EF1/hG-CSF or an EF1/Luc–EF1/hG-CSF expression vector or LRS (control) at indicated times. Differences between EF1/hG-CSF vector and EF1/Luc–EF1/hG-CSF vector are significant (P < 0.05) by t test at days 1, 22, and 29. hG-CSF levels in sera from untreated control mice, as well as from mock-treated control mice (mice receiving HEDGES-mAb cDNAs), were undetectable in this hG-CSF ELISA. (B) Neutrophil counts are displayed from groups of mice treated as in (A). Differences are significant (P < 0.01) at days 8, 15, and 22. (C) Serum hG-CSF levels from groups of mice (five per group) injected with the following DNA/liposome combinations: mCMVenh:hCMVpro/hG-CSF and MLV, mCMVenh:EF1pro/hG-CSF and SUV, and hCMVenh:hCMVpro/hG-CSF and MLV. Differences between MLV and SUV groups subsequently injected with EF1/hG-CSF are significant at days 154, 161, 182, and 197. (D) Hematoxylin and eosin–stained spleen (top) and bone marrow (bottom) tissue sections harvested from control mice or mice injected 582 days earlier with SUV liposomes and EF1/hG-CSF. Scale bars, 25 μm. (E) hG-CSF serum levels from groups of mice (n = 3) injected 24 hours earlier with EF1/hG-CSF plasmid DNA or PCR-generated (linear, closed-end linear, or circularized) EF1/hG-CSF DNA. (F) hG-CSF serum levels at indicated time points from groups of mice (n = 3) injected with EF1/hG-CSF either as plasmid DNA or as PCR-generated circularized DNA. A third group, initially injected with circularized PCR DNA underwent reinjection 35 days after the initial injection. Reinjected PCR and plasmid groups are significantly different at day 106. (A, B, C, E and F) Graphs represent mean ± SEM. One representative result from two to five independent experiments is shown. Statistical significance was tested by the two-tailed Student’s t test or ANOVA where appropriate.

  • Fig. 2 Evolving HEDGES platform incrementally lowers toxicity while increasing expression.

    (A) Alanine transaminase (ALT) and aspartate transaminase (AST) levels at 24 and 48 hours from mice injected with LRS (control) or 1000 nmol of DOTAP liposomes, followed by 100 or 120 μg of an EF1pro/hG-CSF DNA vector. Means ± SEM are displayed. **P < 0.01 compared to control by ANOVA. ns, not significant. No mortality was observed in HEDGES-injected mice receiving up to a 120-μg dose of DNA. (B) hG-CSF and (C) rituximab serum levels 24 hours after HEDGES injection. DOTAP only received 1120 nmol DOTAP liposomes; DOTAP, neutral lipid, and Dex received 1120 nmol DOTAP liposomes incorporating 2.5 mol % Dex-Palm, 1000 nmol DMPC liposomes incorporating 5 mol % Dex-Palm, and intraperitoneal water-soluble dexamethasone (40 mg/kg) 2 hours before intravenous injections. Mice receiving liposomes also received 88 μg of DNA vector encoding hG-CSF or rituximab. (D) Serum ALT levels from groups of mice in (B) and (C) 24 hours after injection. *P < 0.05 and **P < 0.01 by the two-tailed unpaired Student’s t test. One representative result from two to three independent experiments is shown.

  • Fig. 3 One intravenous HEDGES injection of multicassette DNA vectors encoding cognate mAb heavy- and light-chain cDNAs produce prolonged bioactive mAb serum proteins.

    On day 0, groups of CD-1 mice (n = 3) were given dexamethasone intraperitoneally 2 hours before intravenous coinjection of 1120 nmol DOTAP + Dex and 1000 nmol DMPC + Dex liposomes and 2 minutes later intravenously, and then 88 μg of mAb heavy- and light-chain cDNA vector intravenously. Control mice received LRS. (A) Mean ± SEM rituximab serum levels at indicated time points were measured by ELISA. mAb levels in sera from untreated control mice, as well as from mock-treated control mice (mice receiving HEDGES encoding unrelated mAb cDNAs), were undetectable in the rituximab, 5J8 mAb, and mepolizumab ELISAs, respectively. (B) Mean ± SEM viability of CD20+ Raji lymphoma target cells is shown following incubation with human plasma and sera from anti-CD20 or control HEDGES-treated mice isolated at indicated time points. Target Raji cells incubated with recombinant rituximab (10 mg/ml) is also presented for comparison. ***P < 0.001 and *P < 0.05 by ANOVA method. (C) Mean ± SEM anti–IL-5/mepolizumab serum levels are shown over time by ELISA. 5J8 anti-H1 IAV mAb serum levels were measured by ELISA (D) and tested in parallel for capacity to neutralize Cal09 H1N1 IAV in a microneutralization assay (MNT) at indicated time points (E). Serum from HEDGES-rituximab–treated mice (control) served as controls at all time points in ELISA and MNT. (F) Mepolizumab and hG-CSF serum levels over time following intravenous HEDGES injection of an 8.8-kb triple-cassette cDNA vector encoding hG-CSF and mepolizumab heavy- and light-chain cDNAs. Graphs represent mean ± SEM. One representative result from two to three independent experiments is shown.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaax0217/DC1

    Fig. S1. HEDGES hG-CSF cDNA injection elicits the production of endogenous mouse anti–hG-CSF antibodies.

    Fig. S2. Intravenous HEDGES injection of the human TP53 cDNA is expressed predominantly in mouse VECs.

    Fig. S3. Intravenous injection of HEDGES vector DNA does not detectably integrate into genomic DNA of injected mice.

    Fig. S4. HEDGES treatment with a β-GAL reporter-containing plasmid results in expression of reporter in CD31+CD45 lung cells in mice.

    Fig. S5. Development of mouse anti-rituximab antibodies suppresses rituximab levels in HEDGES-treated mice.

    Table S1. HEDGES expression plasmids.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. HEDGES hG-CSF cDNA injection elicits the production of endogenous mouse anti–hG-CSF antibodies.
    • Fig. S2. Intravenous HEDGES injection of the human TP53 cDNA is expressed predominantly in mouse VECs.
    • Fig. S3. Intravenous injection of HEDGES vector DNA does not detectably integrate into genomic DNA of injected mice.
    • Fig. S4. HEDGES treatment with a β-GAL reporter-containing plasmid results in expression of reporter in CD31+CD45 lung cells in mice.
    • Fig. S5. Development of mouse anti-rituximab antibodies suppresses rituximab levels in HEDGES-treated mice.
    • Table S1. HEDGES expression plasmids.

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