Nichtvirale Gentransfersysteme zur Tumortherapie in verschiedenen Mausmodellen

The advantages of non-viral gene transfer systems are safety and
low immunogenicity, therefore they are well suited for use as
vectors in gene therapy. The main disadvantage, namely their low
gene-transfer-efficiency, can be improved through the development
of systemic gene transfer systems using targeted vectors with high
specificity and gene transfer efficiency. The intravenous
application of PEI22lin/DNS complexes leads to a high gene
expression in the lung, but with high toxicity. This observation
can be explained by the positive surface charge of the DNS
complexes and the uncomplexed free PEI, which leads to aggregation
of erythrocytes. DNS complexes can be isolated from free
uncomplexed PEI by gel filtration. The systemic application of gel
filtrated PEI22lin complexes to non-tumor bearing mice resulted in
reduced toxicity however there was a decreased in gene expression
compared to non-filtrated complexes. The same experiment was
performed on tumor bearing mice and again reduced toxicity was
observed and interestingly slightly higher gene expression found in
the tumor compared to the non-filtrated complexes. Shielding the
positive surface charge of the PEI22lin complexes by transferrin
led to increased gene expression in the tumor with reduced
expression in the lung and other organs. The improved tumor
targeted gene expression was associated with reduced systemic
toxicity. Tumor targeted gene expression appears to be dependent on
the tumor model as this observation was only found in neuro2A
neuroblastoma tumor model in A/J mice and not in B16F10 melanoma
tumor models of C57BL/6 mice and CT26 colon carcinoma tumor models
of BALB/c mice. To enhance the intracellular efficiency of the
vectors, the endosomolytic active peptide melittin was incorporated
into the transferrin targeted complexes. This led to a further
increase in gene expression in the Neuro2A tumor models in A/J
mice. For the local gene transfer, electroporation proved to be an
easy to handle method to obtain a high gene expression in tissue.
The non-invasive kaliper electrode was suitable for gene transfer
to both muscle and tumor. The applied voltage showed to be the most
important parameter in expression. The use of electroporation for
intratumoral transfer of the therapeutic gene encoding for the
cytokine TNF was unsuccessful. However, systemic application of
the TNF-α gene in transferrin targeted complexes in combination
with the intraperitoneal application of the chemotherapeutic Doxil
showed a clear synergistic effect. A significant delay in the tumor
growth and in some cases a complete regression of the tumor was
observed. The enzyme cytochrome P450 metabolizes the non toxic
prodrug cyclophosphamide (CPA) into the cytotoxic drug.
Electroporation of the cytochrome P450 gene into the tumor lead to
its localized protein expression. When followed by the
intraperitoneal application of CPA, a significant delay in the
tumor growth of the human hepatocellular carcinoma Huh7 was
observed in SCID mice. When applied to the Neuro2A tumor model in
A/J mice, this application scheme showed a complete tumor
regression in two animals. Furthermore the systemic application of
the P450 gene in transferrin targeted complexes containing melittin
in combination with CPA led to a strong delay in the tumor growth.
In summary, this work describes a new anti-cancer strategy using
the combination of chemotherapeutics and non-viral gene delivery
resulting in a synergistic therapeutic effect in vivo. This
promising strategy will be more effective with the improvement of
non-viral gene delivery systems which have better targeted gene
expression with lower toxicity.