Polymers as gene delivery vehicles gained great success for cancer gene therapy over the last years. At the same time, the sodium iodide symporter (NIS) emerged as outstanding therapy gene. The combinatorial “theranostic” function as a reporter and therapy gene, based on the capacity to transport various radionuclides into the cell, allows for exact quantification of NIS gene expression in the tumor lesion by multimodal imaging of radionuclide uptake as well as therapeutic intervention by application of cytotoxic radionuclides. However, one of the biggest obstacles for nonviral polymer-mediated NIS gene therapy is the design of safe, specific and efficient carriers.
The innovative concept of active receptor-targeted nonviral NIS gene delivery owns enormous potential with high clinical relevance for systemic treatment of solid tumors as well as metastases. But despite the current standard, major progress must be made in the further development of the polymeric delivery systems to improve efficacy, in particular in heterogeneic tumors, biocompatibility, safety, selectivity, uptake and endosomal escape. In the course of this thesis, novel tumor-targeted systems were established and applied for image-guided NIS gene therapy.
The first step was, to set up a broad repertoire of highly efficient and tumor specific ligands.
This enables application of the ligand-mediated NIS gene delivery in a broad range of malignancies that exhibit different receptor expression profiles. The peptide ligands B6 (high tumor-cell binding affinity), GE11 (targeting EGFR) and cMBP (targeting cMET) were coupled to the well-established LPEI-PEG2kDa backbone, which had already been verified as advantageous system with improved performance in vivo. B6-conjugated nonviral delivery vehicles specifically introduce NIS to cancer tissue with high efficacy after systemic application, which was assessed by 123I gamma-camera imaging. In a therapy trial, significant NIS-mediated 131I accumulation induced decelerated tumor growth with prolonged animal survival.
As a next step, EGFR-targeted LPEI-PEG2kDa polyplexes were applied in a colon cancer metastases model. This valuable tool was selected to reflect the clinical situation on a morphological and molecular level including the natural tumor microenvironment and tumor stroma to allow a better prediction of therapy outcome. Using the novel NIS PET-tracer 18 F-TFB, specific imaging of NIS gene expression in single metastases was obtained. Images demonstrated high resolution with exact mapping of metastases that showed significantly increased tracer uptake after EGFR-targeting compared to animals treated with untargeted control polyplexes. Subsequent evaluation of the therapeutic efficacy of the EGFR-targeted
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NIS gene delivery approach resulted in reduced hepatic metastases load of animals treated with 131I, monitored by contrast-enhanced ultrasound, and prolonged animal survival.
With respect to a future translation to humans, where tumor heterogeneity displays a major hurdle causing therapy resistance and limited efficacy, a combinatorial ligand set-up was applied to cope with variable receptor expression levels. Simultaneous targeting of EGFR and cMET for NIS gene therapy combined two outstanding bispecific approaches: (1) application of NIS with its innate dual characteristics to function as reporter and therapy gene and (2) using the synergy of dual-targeting of two receptors. Enhanced uptake and increased NIS gene expression in vitro and in vivo highlighted the benefits of the bifunctional strategy.
Therapeutic intervention by application of cytotoxic 131I resulted in efficient cancer treatment mirrored by reduced tumor growth and improved animal survival and allows successful application even in heterogeneic tumors.
To further improve applicability of delivery vectors, solid-phase methodology was applied for synthesis and design of novel functionalized nanosystems that contain precise numbers of molecules with structure-activity relationship. The precise synthesis allowed for integration of DNA binding sites, shielding domains, amino acids with high buffering capacity and specific ligands that resulted in optimized transfection efficiency, while adverse effects such as toxicity or high immunogenicity were reduced. Based on high cMET expression level on many cancer cells, this receptor serves as an ideal target for cancer therapy. Coupling novel sequence defined polymers to a cMET-binding peptide ligand, systemic NIS gene delivery led to significant tumoral NIS mediated iodide uptake that was sufficiently high for a potent therapeutic effect after 131I application.
To optimize particle structure and performance of shielded PEGylated vectors, a postintegration strategy was conducted. Preformed oleic acid containing sequence defined oligomers were complexed with luciferase or NIS pDNA. As a second step, 1-arm and 2-arm structures composed of PEG coupled to the EGFR-specific peptide ligand GE11 were added to the preformed lipopolyplexes. Chemical characteristics and transfection efficiency were determined in vitro and superiority of 2-arm structures was detected. This strategy forms the basis for a future application in vivo with the advantage of exact formed lipopolyplexes were the effect on size and looser compaction of the shielding domain can be reduced and hydrophobic ligands can be introduced.
In conclusion, in the course of this thesis, the concept of active receptor targeted nonviral NIS gene therapy has been refined. High efficacy and tumor specificity in advanced tumor models was obtained and together with further development of LPEI-based delivery vectors to defined systems, a more efficient and safe application of the image-guided NIS gene therapy was performed. This highlights the enormous potential of nonviral polymeric systems
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as gene transfer systems as well as the outstanding property of NIS in cancer therapy and forms the basis for successful future clinical translation of the NIS gene therapy concept.
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