Targeted TMC-based non-viral vectors for BDNF neuroprotective gene therapy in nerve injury

摘要

Introduction: Peripheral neuropathies may be caused either by diseases of or trauma to peripheral nerves or arise as a consequence of a systemic illness. Conventional treatments offered to manage peripheral neuropathies have primarily been palliative rather than curative. Perhaps most importantly, they have often been ineffective. Envisaging an intervention in peripheral neuropathies it is important to enhance nerve regeneration as well as prevent nerve degeneration. Neurotrophic factors play a crucial role in promoting neuronal trophic support and survival what make them promising disease modulating therapeutic agents to be used in the development of such therapeutic interventions. In this work we present the development of a targeted, non-toxic and efficient carrier for brain-derived neurotrophic factor (BDNF) gene delivery to peripheral neurons in order to efficiently promote neuroprotection/regeneration. The novelty relies on the quaternization of chitosan to improve its transfection ability under physiological conditions and the subsequent functionalization of the DNA polyplexes with a targeting moiety that will confer specificity and the possibility to attain a peripheral administration. Materials and Methods: Trimethyl chitosan (TMC; Mn=43 kDa, DA=11% and quaternization degree of 30%) was used to prepare TMC:DNA complexes. TMC was further modified with thiol groups to prepare neuron-targeted nanoparticles. The 50 kDa non-toxic carboxylic fragment of the tetanus toxin (HC, known as neurotropic and able to be retrogradely transported along axons) was grafted to the binary complex via a bi-functional polyfethylene glycol) (PEG) linker reactive for the thiol moieties in the polymer. We further investigated whether enhanced expression of BDNF by a peripheral intramuscular administration of these vectors could protect sensorial and spinal motor neurons in a sciatic nerve crush injury animal model. Results and Discussion: TMC complexes present higher stability under physiological conditions than CH complexes, resulting in decreased average sizes, lower polydispersion and higher DNA condensation ability. The uptake of the targeted nanoparticles significantly decreased in non-neuronal in relation to neuronal cell lines, indicating an increased specificity to neuronal cells. Our results demonstrate a positive effect of BDNF treatment in the sensorimotor functional recovery as well as a higher expression of neurofilament and Schwann cell markers in the injured nerves of BDNF-treated animals. Besides neuroprotection and neuroregeneration improvement we also observed that BDNF treatment resulted in gastrocnemius muscle protection to denervation that can be responsible for the functional effects. Altogether, our data show the potential of TMC-based vectors to be used as non-viral gene carriers to deliver therapeutic genes to peripheral neurons and thus provide an effective therapeutic intervention for peripheral neuropathies.