The ability to alter cellular behavior via modulation of specific gene expression has great potential in molecular therapeutics, functional genomics and tissue engineering. Several thematically linked approaches from molecular biology, including the use of antisense oligonucleotides (AONs) or short interfering (siRNA), may be used to silence the expression of a target gene. While these are powerful tools that are widely adopted in the research laboratory, the development of more effective delivery systems are sorely needed to add value to the in vitro transfection market and to enable successful application in vivo and in human therapeutics.

The size and charge of nucleic acids preclude their delivery across cellular membranes; furthermore, they are subject to nuclease attack in serum and within certain compartments of the cell. As such, delivery vectors are needed that can protect the oligonucleotides from attack, carry them to and mediate entry into the cells of interest, route them to the site of action (cytoplasm for siRNA and cytoplasm/nucleus for AONs) and release them at this point. We have developed a variety of approaches to accomplish this daunting task, including a cellular-level pharmacodynamic model for the interpretation of results and comparison among various strategies.

Among the delivery systems that we have explored, a most promising one centers around the pH-sensitive polymer, poly(propylacrylic acid, PPAA. This polymer is anionic at physiological pH, allowing it to moderate the charge on lipoplexes between DOTAP and AON as a ternary component to the delivery system. At endosomal pH, PPAA is neutralized, and its hydrophobic character emerges, mediating penetration of cellular membranes. The DOTAP/PPAA/AON (or siRNA) delivery system is quite effective in multiple cell types in the absence of serum, allowing release of oligonucleotide from endosomes which can be visualized throughout the cell and which results in efficient gene silencing. However, it is significantly less effective in the presence of serum.

To improve the performance of the delivery system, we grafted poly(alkylene oxide) moieties onto the backbone of PPAA. We found that a graft using a commercial Jeffamine polymer, which is a copolymer of ethylene oxide and propylene oxide groups, improves cellular delivery and gene silencing markedly under serum-containing conditions where other delivery systems prove ineffective. Effective gene silencing with the DOTAP/PPAA-Jeffamine combination has been observed using either AONs or siRNAs as the cargo, in multiple cell types, and using either GFP as a quantifiable model target or bcl-2 as an oncogenic target. It is interesting to note that at the graft level employed (20%), the PPAA loses much of its pH-dependent hemolytic capacity, but it appears that this is compensated by membrane activity of the Jeffamine polymer. This and other mechanistic questions will be discussed, along with early efforts to apply the system to silence cell resistance pathways in cancer chemotherapy.

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