Zytokin-vermitteltes Targeting von Adenovirusvektoren zur effizienten Transduktion hematopoietischer Zellen
Final Report Abstract
Adenovirus vectors are the most frequently used vector type in clinical trials up to date. However, research in the past has shown that robust targeting strategies need to be developed to render adenovirus vectors suitable tools for gene transfer to Ad-refractory cells like hematopoietic stem and progenitor cells. In addition, after application in vivo, Ad vectors are quickly eliminated by a plethora of vector-host interactions, most of which have not been described in detail. Consistently, shielding and retargeting strategies are mandatory to enable for efficient in vivo gene delivery with this vector type. The aim of our contribution in the priority program SPP1230 was to provide deeper understanding of the molecular basis of successful targeting approaches with a strong focus on post-receptor restrictions. This knowledge should be used to generate adenovirus vectors for improved transduction of human hematopoietic stem and progenitor cells. In addition, we intended to refine existing shielding strategies with the goal to evade vector-host interactions while limiting post-receptor restrictions typically imposed by dense shields. By combining genetic and chemical capsid modifications we could demonstrate that ligand position, ligand size, and intracellular fate of the ligand are critical determinants for the success of Ad vector targeting in vitro and in vivo. Using live cell imaging and advanced fluorescent labelling technologies we showed that impaired nuclear trafficking is the most important post-receptor restriction for (re-)targeted Ad vectors. We set up strategies based on bioresponsive disulfide or hydrazone bonds for the attachment of ligands or shielding moieties to adenovirus capsids that allowed us to overcome this restriction by enabling a separation of ligand/shielding moiety and the vector particle after cell entry. These stratgies may serve as paradigms for a rational development of improved adenovirus gene transfer vectors. Our shielding strategies have revealed that evasion from Kupffer cells – one of the dominant barriers for Ad vectors after in vivo delivery into the blood stream – is feasible. Our data demonstrated that the adenovirus hexon hypervariable region 5 is involved in scavenging and thus provided a molecular basis for improved vector design. These data revealed that position-specific shielding can be used to identify the molecular basis of vector-host interactions and thus can serve as a tool to provide deeper understanding of vector limitations for gene therapy. Finally, we developed a dendrimer-based system that allowed for efficient targeting of Ad5-based vectors to primary human CD34+ hematopoietic progenitor cells in vitro. Using this system transduction efficiencies of >40% could be reached in the absence of toxicity. Of note, cytokines like SCF, IL-3, or GM-CSF that we tested as an alternative strategy were not suitable as ligands for targeting of Ad vectors to hematopoietic progenitor cells.
Publications
- Capsomer-specific fluorescent labeling of adenoviral vector particles allows for detailed analysis of intracellular particle trafficking and the performance of bioresponsive bonds for vector capsid modifications. Hum Gene Ther. 2010 Sep;21(9):1155-67
Espenlaub S, Corjon S, Engler T, Fella C, Ogris M, Wagner E, Kochanek S, Kreppel F
(See online at https://doi.org/10.1089/hum.2009.171) - Fully detargeted polyethylene glycol-coated adenovirus vectors are potent genetic vaccines and escape from pre-existing anti-adenovirus antibodies. Mol Ther. 2008 Jan;16(1):154-62
Wortmann A, Vöhringer S, Engler T, Corjon S, Schirmbeck R, Reimann J, Kochanek S, Kreppel F
(See online at https://doi.org/10.1038/sj.mt.6300306) - Modification of adenovirus gene transfer vectors with synthetic polymers: a scientific review and technical guide. Mol Ther. 2008 Jan;16(1):16-29
Kreppel F, Kochanek S
(See online at https://doi.org/10.1038/sj.mt.6300321) - Reductive amination as a strategy to reduce adenovirus vector promiscuity by chemical capsid modification with large polysaccharides. J Gene Med. 2008 Dec;10(12):1303-14
Espenlaub S, Wortmann A, Engler T, Corjon S, Kochanek S, Kreppel F
(See online at https://doi.org/10.1002/jgm.1262) - Targeting of adenovirus vectors to the LRP receptor family with the high-affinity ligand RAP via combined genetic and chemical modification of the pIX capsomere. Mol Ther. 2008 Nov;16(11):1813-24
Corjon S, Wortmann A, Engler T, van Rooijen N, Kochanek S, Kreppel F
(See online at https://doi.org/10.1038/mt.2008.174) - Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells. Mol Ther. 2011 Jan;19(1):83-92
Prill JM, Espenlaub S, Samen U, Engler T, Schmidt E, Vetrini F, Rosewell A, Grove N, Palmer D, Ng P, Kochanek S, Kreppel F
(See online at https://doi.org/10.1038/mt.2010.229) - Adenoviral vectors coated with PAMAM dendrimer conjugates allow CAR independent virus uptake and targeting to the EGF receptor. Mol Pharm. 2013 Feb 4;10(2):60618
Vetter A, Virdi KS, Espenlaub S, Rödl W, Wagner E, Holm PS, Scheu C, Kreppel F, Spitzweg C, Ogris M
(See online at https://doi.org/10.1021/mp300366f)
