Improved nonviral gene vectors: Efficient and non-toxic polyplexes with enhanced endosomolytic activity

For the development of improved polyethylenimine (PEI) polyplexes
towards ‘artificial viruses’ two key issues are i) to improve the
toxicity profile of the applied vectors and ii) to enhance
endosomal release, one of the major barriers to efficient gene
transfer with PEI polyplexes. Nonviral vectors based on PEI usually
contain an excess of PEI that is not complexed to DNA. Since free
PEI contributes to cellular and systemic toxicity purification of
polyplexes from unbound PEI is highly desirable. In this thesis an
easy and efficient method based on size exclusion chromatography
(SEC) was developed, which for the first time allowed complete
removal of PEI from PEI polyplexes. Moreover, purification of
polyplexes enabled to clarify the role of free PEI in gene delivery
at the cellular level. Most importantly, the removal of unbound PEI
significantly reduced toxicity of the applied polyplexes. However,
purified polyplexes without free PEI were less efficient in
transfection compared to non-purified polyplexes. Mechanistic
studies showed that free PEI most likely enhanced endosomal release
of polyplexes and therefore contributed to efficient gene transfer
with PEI polyplexes. Nevertheless, the availability of a defined,
well-tolerated gene transfer formulation is a vital precondition
for the further development of nonviral gene therapeutics, and a
purification method like the one developed in this thesis will help
to fulfill these requirements. To enhance endosomal release of PEI
polyplexes, the membrane active peptide melittin was incorporated
into the vector particles. It has been shown previously that PEI
bound to the N-terminus of natural all-(L)-melittin
(all-(L)-N-mel-PEI) enhanced gene delivery with PEI polyplexes.
Here, it was demonstrated that transfection efficiency of N-mel-PEI
is independent of the enantiomeric configuration of the bound
peptide which allowed the use of non-immunogenic all-(D)-melittin
for the generation of optimized melittin-PEI conjugates. To
determine the optimal site of melittin-linkage to PEI, the
polycation PEI was covalently attached to the N-terminus
(N-mel-PEI) or the C-terminus of melittin (C-mel-PEI) in
all-(D)-configuration. The site of melittin-linkage had a strong
impact on the membrane destabilizing activities of the resulting
melittin-PEI conjugates. C-mel-PEI was highly lytic at neutral pH
and therefore elevated doses of C-mel-PEI polyplexes induced high
toxicity. In contrast, N-mel-PEI was less lytic at neutral pH but
retained higher lytic activity than C-mel-PEI at endosomal pH 5.
This apparently promoted better endosomal release of N-mel-PEI
polyplexes resulting in efficient gene delivery in different cell
lines. The high potency of C-mel-PEI to destabilize membranes at
neutral pH is presumably due to a reported destabilization
mechanism proceeding through membrane insertion of the peptide. In
contrast, N-mel-PEI is supposed to induce lysis by
insertion-independent pore formation according to the toroidal pore
model. Since membrane destabilization by membrane insertion
requires lower peptide to lipid ratios than destabilization by pore
formation, C-mel-PEI was considered as the more potent template to
generate improved endosomolytic melittin-PEI conjugates. The new
melittin-PEI conjugates should display pronounced lytic activities
at endosomal pH 5. Therefore, PEI was attached to the C-terminus of
melittin analogs which were modified with acidic residues. The
conjugates with the highest lytic activities at endosomal pH 5 were
indeed the most efficient in transfection. This apparent
correlation of gene transfer efficiency with lytic activity at pH 5
was in excellent agreement with results obtained with unmodified
melittin-PEI conjugates and other membrane-active peptides used in
gene transfer. The most efficient melittin-PEI conjugates were
incorporated into surface-shielded and receptor-targeted PEI
polyplexes, and the resulting particles were further purified by
SEC to remove unbound toxic polycations. Endosomolytic melittin-PEI
conjugates stably incorporated into such purified polyplexes
significantly enhanced transfection efficiency in comparison to
polyplexes lacking melittin. Most importantly, these polyplexes
exposed an improved toxicity profile providing an artificial
virus-like vector that is efficient and safe also for potential in
vivo administration.