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Mannose Polyethylenimine Conjugates for Targeted DNA Delivery into Dendritic Cells*

  • Sandra S. Diebold
    Affiliations
    Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13092 Berlin, Germany,
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  • Margaretha Kursa
    Affiliations
    Boehringer Ingelheim Austria, Dr. Boehringer-Gasse 5–11, A-1121 Vienna, Austria, and the
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  • Ernst Wagner
    Affiliations
    Boehringer Ingelheim Austria, Dr. Boehringer-Gasse 5–11, A-1121 Vienna, Austria, and the
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  • Matt Cotten
    Affiliations
    Institute for Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria
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  • Martin Zenke
    Correspondence
    To whom correspondence should be addressed: Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13092 Berlin, Germany. Tel.: 49-30-9406-3343; Fax: 49-30-9406-3329;
    Affiliations
    Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13092 Berlin, Germany,
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  • Author Footnotes
    * This work was supported in part by Deutsche Forschungsgemeinschaft Grant SFB 506 (to M. Z.) and by a grant of the Max-Delbrück-Center Gene Therapy Program (to S. S. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:July 02, 1999DOI:https://doi.org/10.1074/jbc.274.27.19087
      Cell surface-bound receptors represent suitable entry sites for gene delivery into cells by receptor-mediated endocytosis. Here we have taken advantage of the mannose receptor that is highly expressed on antigen-presenting dendritic cells for targeted gene transfer by employing mannosylpolyethylenimine (ManPEI) conjugates. Several ManPEI conjugates were synthesized and used for formation of ManPEI/DNA transfection complexes. Conjugates differed in the linker between mannose and polyethylenimine (PEI) and in the size of the PEI moiety. We demonstrate that ManPEI transfection is effective in delivering DNA into mannose receptor-expressing cells. Uptake of ManPEI/DNA complexes is receptor-specific, since DNA delivery can be competed with mannosylated albumin. Additionally, incorporation of adenovirus particles into transfection complexes effectively enhances transgene expression. This is particularly important for primary immunocompetent dendritic cells. It is demonstrated here that dendritic cells transfected with ManPEI/DNA complexes containing adenovirus particles are effective in activating T cells of T cell receptor transgenic mice in an antigen-specific fashion.
      pL
      polylysine
      TfpL
      transferrin polylysine
      PEI
      polyethylenimine
      TfPEI
      transferrin polyethylenimine
      DC
      dendritic cell(s)
      ManPEI
      mannosylated polyethylenimine
      MHC
      major histocompatibility complex
      GM-CSF
      granulocyte macrophage-colony stimulating factor
      IL
      interleukin
      GFP
      green fluorescent protein
      OVA
      ovalbumin
      Ad
      adenovirus
      wtAd
      wild type Ad
      ManBSA
      mannosylated bovine serum albumin
      BSA
      bovine serum albumin
      PI
      propidium iodide
      FITC
      fluorescein isothiocyanate
      Targeted gene delivery capitalizes on the presence of specific cell surface receptors for DNA uptake into cells by receptor-mediated endocytosis (
      • Cotten M.
      • Wagner E.
      ,
      • Perales J.C.
      • Ferkol T.
      • Molas M.
      • Hanson R.W.
      ,
      • Wu G.Y.
      • Wu C.H.
      ). Therefore, receptor binding ligands are coupled to polycationic compounds like polylysine (pL)1 that bind and condense DNA. Following this concept, transferrin polylysine (TfpL)-based gene transfer systems were developed to target transferrin receptor for DNA delivery into cells (
      • Cotten M.
      • Wagner E.
      ,
      • Cotten M.
      • Wagner E.
      • Birnstiel M.L.
      ,
      • Wagner E.
      • Zenke M.
      • Cotten M.
      • Beug H.
      • Birnstiel M.L.
      ,
      • Zenke M.
      • Steinlein P.
      • Wagner E.
      • Cotten M.
      • Beug H.
      • Birnstiel M.L.
      ,
      • Cotten M.
      • Langle-Rouault F.
      • Kirlappos H.
      • Wagner E.
      • Mechtler K.
      • Zenke M.
      • Beug H.
      • Birnstiel M.L.
      ). Binding of TfpL/DNA complexes to transferrin receptor causes internalization and DNA uptake into the endosomal compartment (
      • Wagner E.
      • Zatloukal K.
      • Cotten M.
      • Kirlappos H.
      • Mechtler K.
      • Curiel D.T.
      • Birnstiel M.L.
      ). To facilitate DNA release from this compartment, endosomolytic agents (such as inactivated adenoviruses) were included in transfection complexes and were demonstrated to effectively enhance gene transfer efficiency (
      • Wagner E.
      • Zatloukal K.
      • Cotten M.
      • Kirlappos H.
      • Mechtler K.
      • Curiel D.T.
      • Birnstiel M.L.
      ,
      • Curiel D.T.
      • Agarwal S.
      • Wagner E.
      • Cotten M.
      ,
      • Cotten M.
      • Wagner E.
      • Zatloukal K.
      • Birnstiel M.L.
      ). More recently, transferrin polyethylenimine (TfPEI) conjugates have been synthesized and used for DNA delivery, thereby combining the high intrinsic transfection efficacy of polyethylenimine (PEI) with receptor-targeted gene transfer (
      • Kircheis R.
      • Kichler A.
      • Wallner G.
      • Kursa M.
      • Ogris M.
      • Felzmann T.
      • Buchberger M.
      • Wagner E.
      ,
      • Diebold S.S.
      • Cotten M.
      • Wagner E.
      • Zenke M.
      ). PEI possesses DNA binding and condensing activity together with a high pH buffering capacity that is believed to protect DNA from degradation and to enhance exit from the endosomal compartment. Accordingly, PEI is effective in gene delivery into a variety of cell types even without the addition of cell binding ligands or endosomolytic agents (
      • Boussif O.
      • Lezoualch F.
      • Zanta M.A.
      • Mergny M.D.
      • Scherman D.
      • Demeneix B.
      • Behr J.P.
      ,
      • Abdallah B.
      • Hassan A.
      • Benoist C.
      • Goula D.
      • Behr J.P.
      • Demeneix B.A.
      ). Here we investigated whether the mannose receptor that is abundantly expressed on dendritic cells (DC) represents a suitable entry site for targeted gene delivery into DC using mannosylated PEI (ManPEI).
      DC are professional antigen-presenting cells that occur in peripheral organs like skin, where these cells are exposed to antigens, which they capture and process (
      • Peters J.H.
      • Gieseler R.
      • Thiele B.
      • Steinbach F.
      ,
      • Cella M.
      • Engering A.
      • Pinet V.
      • Pieters J.
      • Lanzavecchia A.
      ,
      • Austyn J.M.
      ,
      • Banchereau J.
      • Steinman R.M.
      ). Upon inflammatory stimuli, DC migrate to lymphoid tissue and present processed antigens on major histocompatibility complex (MHC) class I and II molecules to T cells, to elicit an antigen-specific T cell response. Because of their central role in the initiation of primary immune responses, there is high interest in employing DC for immunotherapy of diseases, such as cancer (
      • Girolomoni G.
      • Ricciardi-Castagnoli P.
      ,
      • Tuting T.
      • Storkus W.J.
      • Lotze M.T.
      ,
      • Schuler G.
      • Steinman R.M.
      ). Following such approaches, gene-modified DC offer several potential advantages over peptide/protein-pulsed DC. For example, gene-modified DC can be expected to induce T cell responses against multiple and/or undefined epitopes of tumor antigens, possibly in the context of both MHC class I and II, and with any MHC allele. Furthermore, the expression of chemokines and cytokines in DC simultaneously with tumor-specific and/or associated antigens would additionally allow modulation of the immune response. DC and T cell functions are effectively regulated by a variety of cytokines, and local cytokine production by DC might represent an important adjunct for T cell activation in medical therapy, for example in cancer patients who are often immunosuppressed. However, so far the generation of gene-modified immunocompetent DC has remained difficult mainly due to limitations in DNA delivery techniques (
      • Diebold S.S.
      • Cotten M.
      • Wagner E.
      • Zenke M.
      ,
      • Schuler G.
      • Steinman R.M.
      ,
      • Arthur J.F.
      • Butterfield L.H.
      • Roth M.D.
      • Bui L.A.
      • Kiertscher S.M.
      • Lau R.
      • Dubinett S.
      • Glaspy J.
      • McBride W.H.
      • Economou J.S.
      ).
      DC express high levels of mannose receptor and mannose receptor-related receptor that are used for endocytosis and phagocytosis of a variety of antigens that expose mannose and fucose residues (
      • Cella M.
      • Engering A.
      • Pinet V.
      • Pieters J.
      • Lanzavecchia A.
      ,
      • Jiang W.
      • Swiggard W.J.
      • Heufler C.
      • Peng M.
      • Mirza A.
      • Steinman R.M.
      • Nussenzweig M.C.
      ,
      • Steinman R.M.
      • Swanson J.
      ,
      • Sallusto F.
      • Cella M.
      • Danieli C.
      • Lanzavecchia A.
      ,
      • Avrameas A.
      • McIlroy D.
      • Hosmalin A.
      • Autran B.
      • Debre P.
      • Monsigny M.
      • Roche A.C.
      • Midoux P.
      ). DC also express transferrin receptor, albeit at lower levels.
      S. S. Diebold and M. Zenke, unpublished observations.
      2S. S. Diebold and M. Zenke, unpublished observations.
      Following ligand binding, internalization, and release of cargo, both mannose and transferrin receptor are recycled and transported back to the cell surface, where they allow repeated internalization of new ligand molecules (
      • Cotten M.
      • Wagner E.
      ,
      • Cotten M.
      • Wagner E.
      • Birnstiel M.L.
      ,
      • Stahl P.D.
      ). For this reason, mannose receptor might be similar to transferrin receptor and particularly suited for targeted delivery of DNA into cells by employing synthetic mannose polycation conjugates and using a strategy that was successfully applied before for transferrin receptor. Here we describe the synthesis of ManPEI conjugates and have investigated uptake of ManPEI/DNA transfection complexes and their efficacy in DNA delivery. Based on previous successful enhancement of receptor-mediated gene delivery by endosomolytic agents, ManPEI/DNA complexes containing endosome-disrupting adenovirus particles were also generated and investigated.

      DISCUSSION

      Here we describe the synthesis of ManPEI conjugates and their analysis for receptor-targeted gene delivery. ManPEI transfection represents a fully synthetic delivery system that capitalizes on gene transfer by receptor-mediated endocytosis via surface-bound mannose receptor that is highly expressed on antigen-presenting DC. By employing ManPEI rather than mannosylated polylysine conjugates, we took advantage of the higher transfection potential of conjugated PEI as observed for transferrin conjugates (TfPEI versus TfpL) in various cell types (
      • Kircheis R.
      • Kichler A.
      • Wallner G.
      • Kursa M.
      • Ogris M.
      • Felzmann T.
      • Buchberger M.
      • Wagner E.
      ) and also in initial TfPEI transfection studies in DC (
      • Diebold S.S.
      • Cotten M.
      • Wagner E.
      • Zenke M.
      ). Mannosylated polylysine conjugates were employed in related studies for targeting mannose receptor in macrophages (
      • Erbacher P.
      • Bousser M.T.
      • Raimond J.
      • Monsigny M.
      • Midoux P.
      • Roche A.C.
      ,
      • Ferkol T.
      • Perales J.C.
      • Mularo F.
      • Hanson R.W.
      ).
      Several ManPEI conjugates were synthesized by reductive amination with mannobiose or by coupling with mannosylphenylisothiocyanate to generate Man-bio-PEI and Man-itc-PEI conjugates, respectively. Both conjugates were found to exhibit similar physical properties and transfection potential when tested in mannose receptor-positive BM2 myeloblasts. The influence of low and high molecular mass PEI (25- and 800-kDa PEI, respectively) on ManPEI/DNA transfection was also studied. Again, ManPEI25 and ManPEI800 conjugates showed similar transfection efficiencies. Additionally, blocking experiments demonstrated that the uptake of ManPEI/DNA transfection complexes was mannose receptor-specific.
      While these studies demonstrated that ManPEI conjugates are effective in gene delivery via mannose receptor, their transfection potential for primary human and mouse DC was found to be rather low. This appears not to be due to differences in mannose receptor levels, which were the same for BM2 cells and mouse DC and even higher in human DC. Importantly, incorporation of adenovirus particles in ManPEI transfection complex dramatically increased transgene expression as observed for both wild type Ad particles as well as replication-deficient E4 Ad particles. This might be due to the fact that, following uptake of such Ad/ManPEI/DNA complexes via mannose receptor, the adenovirus component facilitates DNA release from the endosomal compartment, similar to its action in adenovirus-augmented transferrinfection (
      • Cotten M.
      • Wagner E.
      • Birnstiel M.L.
      ,
      • Wagner E.
      • Zatloukal K.
      • Cotten M.
      • Kirlappos H.
      • Mechtler K.
      • Curiel D.T.
      • Birnstiel M.L.
      ,
      • Curiel D.T.
      • Agarwal S.
      • Wagner E.
      • Cotten M.
      ,
      • Cotten M.
      • Wagner E.
      • Zatloukal K.
      • Birnstiel M.L.
      ). Alternatively adenovirus itself might contribute to uptake of Ad/ManPEI/DNA complexes via the adenovirus infection route. Such an idea would be in line with the finding that Ad/PEI/DNA transfection complexes containing plasmid DNA bound to adenovirus carrier via PEI (
      • Baker A.
      • Saltik M.
      • Lehrmann H.
      • Killisch I.
      • Mautner V.
      • Lamm G.
      • Christofori G.
      • Cotten M.
      ,
      • Baker A.
      • Cotten M.
      ,
      • Fasbender A.
      • Zabner J.
      • Chillon M.
      • Moninger T.O.
      • Puga A.P.
      • Davidson B.L.
      • Welsh M.J.
      ) are effective in delivering genes into DC (
      • Diebold S.S.
      • Lehrmann H.
      • Kursa M.
      • Wagner E.
      • Cotten M.
      • Zenke M.
      ). Furthermore, Ad/ManPEI/DNA transfection complexes containing a high number of adenovirus particles were found to be less affected by blocking the mannose receptor internalization route with ManBSA than complexes not containing adenovirus particles. Thus, Ad/ManPEI/DNA complexes apparently bind to cells and deliver DNA at least in part via the adenovirus moiety of the complex.
      It appears therefore that in DC uptake of DNA via the adenovirus internalization route is more effective than via mannose receptor, while for BM2 cells both routes are equally efficient. In DC, the difference between both uptake pathways might be the extent of endosomal degradation that could be particularly high for mannose receptor-targeted complexes. We have attempted to address this question by applying agents that increase the endosomal pH and thereby inhibit lysosomal degradation like chloroquin and monensin (
      • Zenke M.
      • Steinlein P.
      • Wagner E.
      • Cotten M.
      • Beug H.
      • Birnstiel M.L.
      ,
      • Cotten M.
      • Langle-Rouault F.
      • Kirlappos H.
      • Wagner E.
      • Mechtler K.
      • Zenke M.
      • Beug H.
      • Birnstiel M.L.
      ,
      • Dean R.T.
      • Jessup W.
      • Roberts C.R.
      ). However, so far these studies have met with only limited success due to the high unspecific cytotoxicity of the compounds used.
      Finally, our study demonstrates that ManPEI/DNA complexes containing adenovirus particles are effective in activating T cells from T cell receptor transgenic mice in an antigen-specific manner. DC transfected with ManPEI/DNA complexes without adenovirus particles were deficient in inducing such a T cell response, which is presumably related to low transgene expression, and higher expression levels might be required to overcome this limitation. Current experiments aim at incorporating synthetic endosome-disruptive influenza peptides (
      • Plank C.
      • Oberhauser B.
      • Mechtler K.
      • Hoch C.
      • Wagner E.
      ) in ManPEI transfection complexes to address this question. Such a system would have the advantage of being fully synthetic.
      The present study opens the possibility to use ManPEI transfection for gene delivery into DC to study DC function and to develop DC-based approaches of immunotherapy, e.g. of cancer or viral or infectious diseases. Other transfection techniques so far tested for DC were mostly inefficient and associated with high unspecific cytotoxicity and low transgene expression (
      • Diebold S.S.
      • Cotten M.
      • Wagner E.
      • Zenke M.
      ,
      • Arthur J.F.
      • Butterfield L.H.
      • Roth M.D.
      • Bui L.A.
      • Kiertscher S.M.
      • Lau R.
      • Dubinett S.
      • Glaspy J.
      • McBride W.H.
      • Economou J.S.
      ). Additionally, DC from peripheral blood monocytes, as used in this study, are largely postmitotic and difficult to infect with recombinant retroviruses that rely on proliferating cells (
      • Aicher A.
      • Westermann J.
      • Cayeux S.
      • Willimsky G.
      • Daemen K.
      • Blankenstein T.
      • Uckert W.
      • Dorken B.
      • Pezzutto A.
      ,
      • Westermann J.
      • Aicher A.
      • Quin Z.
      • Cayeux S.
      • Daemen K.
      • Blankenstein T.
      • Dörken B.
      • Pezzutto A.
      ). More recently recombinant adenovirus vectors were applied for transduction of DC (
      • Diebold S.S.
      • Cotten M.
      • Wagner E.
      • Zenke M.
      ,
      • Arthur J.F.
      • Butterfield L.H.
      • Roth M.D.
      • Bui L.A.
      • Kiertscher S.M.
      • Lau R.
      • Dubinett S.
      • Glaspy J.
      • McBride W.H.
      • Economou J.S.
      ,
      • Ribas A.
      • Butterfield L.H.
      • McBride W.H.
      • Jilani S.M.
      • Bui L.A.
      • Vollmer C.M.
      • Lau R.
      • Dissette V.B.
      • Hu B.
      • Chen A.Y.
      • Glaspy J.A.
      • Economou J.S.
      ,
      • Song W.
      • Kong H.L.
      • Carpenter H.
      • Torii H.
      • Granstein R.
      • Rafii S.
      • Moore M.A.S.
      • Crystal R.G.
      ). The ManPEI transfection system described in this paper is particularly versatile and offers several advantages over viral vectors. For example, very large DNA constructs (more than 100 kilobase pairs) can be transfected to ensure long lasting transgene expression (
      • Baker A.
      • Saltik M.
      • Lehrmann H.
      • Killisch I.
      • Mautner V.
      • Lamm G.
      • Christofori G.
      • Cotten M.
      ). More importantly, several plasmid DNAs can be transfected simultaneously to induce and/or modulate immune responses. Current experiments address the question of whether such a modulation of the immune response with gene-modified DC can be achieved by coexpression of the antigen with specific cytokines and chemokines that activate or attract T cells.

      Acknowledgments

      We thank Novartis (Vienna, Austria) and Schering-Plough, (Kenilworth) for recombinant human GM-CSF and IL-4, respectively. We are most grateful to R. Holzhauser for conjugate synthesis; F. R. Carbone, M. Lutz, and G. Schuler for OT-I mice; T. Pezzutto and J. Westermann for plasmid DNA; and S. M. Kurz for recombinant mouse GM-CSF. We thank T. Blankenstein for helpful discussions, T. Schüler for advice in T cell preparation, C. Esslinger for careful reading of the manuscript, and I. Gallagher for expert secretarial assistance.

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