Novel biodegradable gene carriers based on oligomerized polyamines

Gene therapy is a very promising approach to treat or to prevent
diseases. However, progress in this field is hindered by lack of
suitable vectors. Current research focuses on the development of
novel nonviral biodegradable gene carriers with improved gene
transfer activity and low toxicity. In the course of this thesis, a
library of degradable DNA compacting domains based on oligomerized
polyamines was synthesized and analyzed. Degradation of the
originated polymers was either based on site-specific reductive
cleavage of disulfide bonds or on time-dependent ester/amide
hydrolysis. DNA binding activity, polyplex stability, transfection
efficiency, toxicity, and hemocompatibility studies were performed
in order to identify promising candidates. Some of the novel gene
carriers, especially the degradable oligoethylenimine (OEI)
derivatives were successfully applied for in vitro transfection and
could easily compete with the current ‘golden standard’ linear
polyethylenimine with an average Mw of 22 kDa (PEI22lin).
Furthermore, screening results revealed critical structure activity
relationships which were very helpful for improving the polymer
design. According to transfection and biocompatibility results,
efficiency and toxicity correlated to some degree. Polymers with an
overall high charge density and a high molecular weight like
OEI-HD-1 provided polyplex stability and formed small uniform
particles. On the other hand these polymers tended to induce
erythrocyte aggregation and exhibited a pronounced cytotoxicity
when applied at high concentrations. Polycation with a lower
molecular weight (~ 10 kDa) like e.g. OEI-IP-1 were essentially
nontoxic, but had to be applied at high concentrations in order to
achieve efficient gene transfer. Intrinsic membrane activity of
certain polymers could damage cellular membranes but may also
trigger endosomal release and therefore boost transfection
activity. Crosslinking of OEI 800 with 1,6-hexanedioldiacrylate
resulted in highly efficient degradable polycations. Different
reaction temperatures during OEI-HD-1 synthesis had a strong impact
on molecular weight and the ester/amide ratio. Despite structural
differences, both OEI-HD-1 (synthesized at 60°C) and lt-OEI-HD-1
(synthesized at 20°C) possessed equal gene transfer activity as the
‘golden standard’ PEI22lin when applied at their optimal
polymer/DNA-ratio (w/w). It was important to note that lt-OEI-HD-1,
the LMW-derivative which is predominantly based on ester linkages,
was significantly less toxic than its HMW amide-linked counterpart.
OEI-HD displayed a very promising basis for the development of
further powerful gene carriers. A two-step synthesis protocol was
established in order to generate OEI-HD cores bearing excessive
linker which could be subsequently modified with various
functionalities like spermine. OEI-HD-Sper pseudo-dendrimers were
characterized by a pronounced intrinsic membrane activity and
possess high transfection efficiency. Since current nonviral
vectors are still very inefficient as compared to their viral
competitors, natural viruses present an ideal example educating us
how to further optimize polycationic gene carriers in terms of
specific cell-targeting and improved endosomal release.
Modification of polyplexes towards a “smart” virus-like system was
achieved in the following way. Degradable DNA compacting domains
(OEI-HD-1) were utilized for complex formation. Furthermore,
epidermal growth factor (EGF) was incorporated as targeting ligand
into OEI-HD-1 polyplexes and thus allowed cell-specific cellular
uptake via the EGF receptor (EGFR). Gene transfer potential of
EGFR-targeted degradable polyplexes was further improved by
applying technologies which promoted the endosomal release of
endocytosed particles. Photochemical intracellular release (PCI) is
based on accumulation of amphiphilic photosensitizers (PS) in
endosomal membranes. Illumination of PS pre-treated transfected
cells results in activation of the PS and subsequent light-induced
rupture of endocytic vesicles. Combination of biological (EGFR) and
physical (PCI) targeting greatly enhanced reporter gene delivery
mediated by OEI-HD-1 polyplexes. Finally, the incorporation of
membrane active melittin derivatives into EGF/OEI-HD-1 polyplexes
was the first example of a biodegradable synthetic virus for gene
delivery.