The ability to modulate cellular behavior through genetic modification has great potential in molecular therapeutics, functional genomics and tissue engineering. For instance, antisense oligonucleotides (AS ONs), which are most commonly single-stranded DNA molecules 15-25 nucleotides in length, modulate gene expression by binding to a complementary segment on a target mRNA, thus repressing translation of the encoded protein. While antisense technology is becoming a viable therapeutic entity and platform for functional genomics, intracellular delivery of the polynucleic acid remains a major barrier to more widespread development. For antisense technology and alternative approaches involving oligonucleotides, such as RNA interference (RNAi), delivery systems are borrowed primarily from those used for non-viral gene delivery, despite the fact that the latter are highly inefficient.

We are developing a molecular approach to improving cellular delivery by controlling the electrostatic interactions and intracellular trafficking involved in cationic lipid and polymer-mediated delivery of AS ODNs. We have developed structure-activity relationships for the delivery of ONs using cationic polymers (e.g., polyethyleneimine) and in particular developed some insights into how antisense activity is manifest in the biophysical properties of the ON (backbone) and polymer (molecular weight, branching). We show that antisense activity can be tuned to varying time scales by subtle changes in molecular properties, and we show how a mixed-backbone oligonucleotide may provide an optimum balance of nuclease stability, polyelectrolyte complexation strength, and avoidance of non-specific binding.

We also show that endosomal release is a major barrier to delivery effectiveness and that a synthetic, pH-sensitive, membrane-disrupting polyanion, poly(propylacrylic acid) (PPAA) improves the in vitro efficiency of the cationic lipid DOTAP with regards to ON delivery and antisense activity. Incorporation of PPAA in DOTAP/ON complexes improves substantially the cellular uptake of fluorescently labeled oligonucleotides and the AS ON activity over a range of PPAA concentrations, particularly in serum-free media. Our current work focuses on extending the applicability of this system.

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