The neurotoxicity of gene vectors and its amelioration by packaging with collagen hollow spheres.

Over the last twenty years there have been several reports on the use of nonviral vectors to facilitate gene transfer in the mammalian brain. Whilst a large emphasis has been placed on vector transfection efficiency, the study of the adverse effects upon the brain, caused by the vectors themselves, remains completely overshadowed. To this end, a study was undertaken to study the tissue response to three commercially available transfection agents in the brain of adult Sprague Dawley rats. The response to these transfection agents was compared to adeno-associated viral vector (AAV), PBS and naked DNA. Furthermore, the use of a collagen hollow sphere (CHS) sustained delivery system was analysed for its ability to reduce striatal toxicity of the most predominantly studied polymer vector, polyethyleneimine (PEI). The size of the gross tissue loss at the injection site was analysed after immunohistochemical staining and was used as an indication of acute toxicity. Polymeric vectors showed similar levels of acute brain toxicity as seen with AAV, and CHS were able to significantly reduce the toxicity of the PEI vector. In addition; the host response to the vectors was measured in terms of reactive astrocytes and microglial cell recruitment. To understand whether this gross tissue loss was caused by the direct toxicity of the vectors themselves an in vitro study on primary astrocytes was conducted. All vectors reduced the viability of the cells which is brought about by direct necrosis and apoptosis. The CHS delivery system reduced cell necrosis in the early stages of post administration. In conclusion, whilst polymeric gene vectors cause acute necrosis, administration in the brain causes adverse effects no worse than that of an AAV vector. Furthermore, packaging the PEI vector with CHS reduces surface charge and direct toxicity without elevating the host response.

The effect of intraluminal contact mediated guidance signals on axonal mismatch during peripheral nerve repair.

The current microsurgical gold standard for repairing long gap nerve injuries is the autograft. Autograft provides a protective environment for repair and a natural internal architecture, which is essential for regeneration. Current clinically approved hollow nerve guidance conduits allow provision of this protective environment; however they fail to provide an essential internal architecture to the regenerating nerve. In the present study both structured and unstructured intraluminal collagen fibres are investigated to assess their ability to enhance conduit mediated nerve repair. This study presents a direct comparison of both structured and unstructured fibres in vivo. The addition of intraluminal guidance structures was shown to significantly decrease axonal dispersion within the conduit and reduced axonal mismatch of distal nerve targets (p < 0.05). The intraluminal fibres were shown to be successfully incorporated into the host regenerative process, acting as a platform for Schwann cell migration and axonal regeneration. Ultimately the fibres were able to provide a platform for nerve regeneration in a long term regeneration study (16 weeks) and facilitated increased guidance of regenerating axons towards their distal nerve targets.