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Photochemical internalization (PCI) technology has been used for PEI-mediated p53 gene transfer in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts. Using luciferase as a reporter gene, PCI led to a 20-fold increase in transgene expression 48 h after transfection and sustained transgene expression for 7 days. Therefore, iterative p53 gene transfer was performed by means of a weekly single injection of PEIGlu4/p53 complexes alone or with PCI for 5 (group A) or 7 (group B) weeks. The efficiency of p53 gene therapy was evaluated by following tumor growth and expression of P53-related downstream proteins (P21, MDM2, Bcl2, Bax). Apoptosis induction was evidenced through caspase-3 activation and PARP cleavage. Using PCI, tumor growth inhibition was observed in all transfected animals. Further, successful tumor cure was achieved in 17% (group A) and 83% (group B) of animals. PCI-mediated p53 gene transfer led to higher P53 protein expression that was correlated with induction of Bax and P21 proapoptotic proteins, repression of Bcl2 as well as activation of caspase-3, and cleavage of PARP. The present study demonstrates that PCI enhances the in vivo efficiency of PEI-mediated p53 gene transfer and can be proposed for p53 gene therapy in HNSCC.

Protein delivery is emerging as an interesting alternative to gene delivery. We have used our experience with transfection to develop a technique for efficient delivery of anionic proteins such as antibodies into the cytoplasm of cells. As for DNA, when complexed with cationic lipids, large amounts of proteins are shown to enter adherent cells via ubiquitously expressed syndecans. However, protein surface area rather than electric charge ratio governs the delivery characteristics. Delivery of anti-beta-actin and anti-alpha-tubulin IgG's leads to fiber depolymerization. Intracellular delivery of an antibody could thus be regarded as another method for interfering with gene activity.

Top of pageAbstract Linear polyethylenimine (L-PEI) is the most used non-viral delivery reagent for gene delivery in animals. Various delivery routes of small size defined PEI polyplexes formulated in a 5% glucose solution can be used (intravenous, intracerebral, intraperitoneal, intracardiac, instillation |[hellip]|). Intravenous injection is the route of choice in many gene therapy applications. Following systemic delivery of PEI polyplexes, a non-specific targeting of lungs occurs where c.a. 90% of transgene expression are found. We have addressed the bolus effect caused with the injection volume of polyplexes solution using jetPEI|[trade]| after the rapid injection (few seconds) into the tail vein of mice. We have shown that injection volume less than 200|[mu]|l leads to lower transgene expression in lungs than volumes comprised between 200 and 400|[mu]|l. When the injection volume of polyplexes is increased to 2-2.5 ml and the injection performed by perfusion, the transgene level is slightly affected in lungs. The optimal effect of bolus is obtained following a rapid injection (c.a. 10 seconds) of 400|[mu]|l of a 5% glucose solution. It is generally demonstrated that highly positive polyplexes (N/P ratio >4) are efficient in the lungs. We have purified polyplexes formulated with various N/P ratios by ultracentrifugation. We have shown that as expected, the amount of free PEI removed increased with the initial N/P ratio used to prepare polyplexes and reached more than 50% at N/P ratio of 10. When the optimal injection conditions were applied with polyplexes made initially with a N/P ratio of 10 and then purified, a 10-fold decrease in gene expression in the lung was observed. Increasing the dose of DNA injected using purified polyplexes increased the transgene level comparable to the optimal conditions obtained with crude complexes. It appears that the presence of free polymer in the optimal crude complexes has a co-helper effect. The role of this free polymer to reach a high transgene level in vivo is not yet identified and needs further investigations.

The recent identification of neural stem cells (NSC) in the subventricular layer of the adult mammalian brain opens up exciting perspectives for therapy of neuro-degenerative diseases. However, the in vivo capacity of neural stem cells to self renew and differentiate are still undefined. A powerful means of investigating the mechanisms regulating NSC biology is RNA interference (RNAi). To exploit the enormous potential of RNAi, efficient in vivo vectorisation methods are needed. Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo. We used the newborn mouse brain as optimisation is easier and because the radial glial cells that give rise to the adult stem cell population are well characterised and likely targeted by the vectors employed.