El mariachi en el Ecuador – Cantón Riobamba

nostructures

The advantages of using DNA nanostructures as carriers for immunoactive oligonucleotides instead of free DNA strands are the increased stability discussed in the previous section, the potential of targeted delivery via functionalization and high local and controllable surface density by precise spatial arrangements of the immune stimulating oligonucleotides on the DNA delivery system [79, 80, 84]. Immunoactive oligonucleotides are short DNA strands of specific sequences that act as artificial stimulants of the immune system by mimicking the DNA sequence pattern of pathogenic microbes. They are therefore a promising agent in vaccination strategies for the therapy of cancer, allergy and infectious diseases [151, 152].

80 nm 20 nm CpG

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Figure 3.3: Immunestimulation by CpG Oligodeoxynucleotides delivered by DNA nanostruc-

tures. ACpG/TLR9-mediated pathway: CpG ODN uptake, TLR9 recognition, cytokine segrega- tion and expression of surface molecules. BHollow DNA origami tube-like structure of publication P2 decorated with 62 CpG ODNs. C Polypod-like DNA structures delivering three (top) and six (bottom) CpG ODNs per nanoparticle. Reprinted with permission from ref. [83]. Copyright 2012 American Chemical Society. D Tetrahedral DNA structure with four CpG ODNs. Reprinted with permission from ref. [79]. Copyright 2011 American Chemical Society. E In vivo immune activation in mice by CpG ODN containing DNA tetrahedron vaccine complexes consisting of CpG ODN adjuvant molecules (purple) and model antigen streptavidin (red). Reprinted with permission from ref. [84]. Copyright 2012 American Chemical Society.

The immune stimulating sequence of the oligonucleotide used in publication P2 is based on the finding by Krieg et al. in 1995. He reported that so-called ’CpG-motifs’, which con- sist of unmethylated cytosine-phosphate-guanine dinucleotides integrated in some specific sequences, are responsible for previously described immune stimulating effects of bacterial DNA on distinct mammalian immune cells [153]. The fact that the genomes of vertebrates contain significantly less CpG motifs than the genomes of microbes supported the hypothesis of selective differentiation by the mammalian immune system between endogenous and inva- sive DNA from microorganisms like bacteria and viruses via specific DNA receptors [153, 154]. One such DNA receptor that mediates the activation of innate immunity is the endosomal Toll-like receptor 9 (TLR9), which recognizes unmethylated CpG sequences [155, 156]. As a consequence of TLR9 stimulation, proinflammatory cytokines, interferons and chemokines are secreted and particular transmembrane proteins are expressed (see figure 3.3 A) [157]. In publication P2, cytokines such as interleukin IL-6 in the supernatant of immune cells and the surface expression of the transmembrane C-type lectin CD69, an early marker of immune activation, are used as parameters to quantify the degree of external immune stimulation. To enhance the delivery of CpG oligodeoxynucleotides (ODNs) into the endosomes of mam- malian immune cells and thus initiate immune responses stronger than with free CpG ODNs, several groups have developed various DNA nanocarriers. In 2008, Takakura et al. developed

a Y-shaped DNA nanostructure consisting of three CpG motifs that provoked significantly higher amounts of IL-6 and tumor necrosis factor-α (TNF-α) in macrophage-like, TLR9- positive RAW263.7 cells than free CpG ODNs due to the increased cellular uptake of the DNA carrier system [77]. Further parameters like the increased local density within the endo- some and the accessibility of the 5’-ends of the CpG motif can be crucial factors that influence this enhancement in immune stimulatory activity [78]. More complex DNA constructs like dendrimer-like and polypod-like structures that deliver higher amounts of CpG ODNs per particle showed further increased efficiency in immune stimulation in RAW264.7 cells (see figure 3.3 C)[78, 83]. In addition, three-dimensional tetrahedral DNA nanostructures were used as delivery system for up to four appending PTO modified CpG motifs, which induced cytokine segregation dependent on the number of CpG ODNs attached to the DNA tetrahe- dron (see figure 3.3 D)[79].

In publication P2, the local surface density could be raised to 62 CpG ODNs per carrier by attaching them onto a hollow DNA origami tube-like structure (see figure 3.3 B)[80]. The ex vivo immune activation by the DNA CpG origami complexes were characterized by IL-6 and IL-12 segregation and CD69 expression of freshly isolated spleen cells from mice. DNA origami bound CpG ODNs caused a significantly enhanced immune response compared to free CpG ODNs, especially when the CpG motifs were PTO modified. To exclude immune activation by the DNA origami structure itself, all parts of the system were tested sepa- rately. Even if a mediate immune response due to undecorated DNA origami structures was detected, CpG carrying complexes provoked considerably higher immune stimulation. DNA origami tubes with a control ODN sequence, where CpG was replaced by GpC and the test on TLR9 deficient mice cells further confirmed the assumed immune stimulation pathway. Liu et al. were the first to demonstratein vivo immune activation in mice after transfection with CpG ODN containing DNA tetrahedra (see figure 3.3 E)[84]. By functionalization of the CpG DNA tetrahedon complex with the model antigen streptavidin, a synthetic vaccine construct was generated that induced strong antibody responses.

To achieve the aim of developing powerful delivery systems like envisioned by Paul Ehrlich and in particular to design immunogenic vaccines, the close spatial arrangement of immune active substances like CpG ODNs to an antigen is crucial. DNA nanostructures can exactly meet this requirement.