Abstract
The sequencing of the human genome was expected to generate a veritable explosion of
therapeutics for regulation of gene function, either through modulation of gene
expression or through the replacement of defect genes. However, nucleic acid-based
pharmacological agents suffer from issues of low bioavailability and unfavorable
pharmacokinetics, wherefore these prospects have not yet been realized.
One promising approach for regulation of gene function is a special type of antisense
technology, referred to as splice correction. Aberrantly spliced mRNA is intimately
associated with numerous serious illnesses, wherefore the ability to restore the correct
splicing pattern is a highly attractive therapeutic approach. Another thriving
oligonucleotide-based platform makes use of small (or short) interfering RNA (siRNA),
double-stranded RNA sequences that efficiently silence expression of essentially any
gene of interest. However, both platforms are limited by the inherent weaknesses of
oligo- and polynucleotide-based agents, meaning that the development of efficient
delivery vectors is a prerequisite for clinical translation. Short cationic peptide
sequences, so called cell-penetrating peptides (CPPs), constitute an emerging category of
delivery vehicles with the ability to convey various cargo molecules across the cell
membrane, but numerous polymeric vectors (commonly referred to as ‘polyplexes’) are
also under intense scrutiny for delivery of gene-regulating agents.
This thesis aims to delineate the internalization mechanisms of CPPs conjugated to a
special type of splice-correcting oligonucleotide analogues (namely peptide nucleic
acids (PNAs)), but it also presents a rationally modified CPP for delivery of splicecorrecting oligonucleotides and plasmid DNA, as well as an entirely novel class of
delivery vectors, so called polythiophenes, for siRNA delivery. Specifically, paper I
examines the internalization routes of a number of CPP-PNA conjugates, papers II and
III study the oligonucleotide and plasmid delivery efficacy, respectively, of the
stearylated CPP transportan 10 (TP10), whereas paper IV examines the utility of a
cationic polythiophene for siRNA delivery.
In conclusion, the research described herein provides novel data on internalization
mechanisms of chemically distinct CPPs, as well as presents two novel agents for
delivery of splice-correcting oligonucleotides, plasmid DNA, and siRNA, thereby
adding additional tools to the toolbox for delivery of gene-regulating agents.
