CONICET | Buscador de Institutos y Recursos Humanos

Gene therapy has undergone a remarkable development in the last 20 years with important advances having been made in the improvement of gene transfer and expression technology. The association of viral vector-based gene delivery with nanotechnology now offers the possibility to develop more efficient and less invasive gene therapy strategies for a number of major pathologies like Parkinson?Ls and Alzheimer?Ls disease. This approach combines Magnetic Drug Targeting (MDT) and magnetofection, two novel methodologies based on the use of magnetic nanoparticles (MNP). The concept of MDT was introduced in 1978 by the group led by K. J. Widder and its goal is to concentrate magnetically responsive therapeutic complexes in target areas of the body by means of external magnetic fields. So far, the main application of MDT has been cancer therapy. Typically, magnetic microparticles (?Êm sized) or MNP (nm sized) associated to a therapeutic drug are intravascularly injected near the tumor blood supply and are concentrated into the tumor by means of an external magnetic field. This strategy has shown promising results in clinical trials. Magnetofection is a methodology developed in the early 2000?fs by the group led by C. Plank in Munich. It is based on the association of MNP with nonviral or viral vectors in order to optimize gene delivery in the presence of a magnetic field. In order to implement magnetofection in the brain we have constructed two adenoviral vectors, RAd-DsRed2 which expresses DsRed2, a red fluorescent protein from Discosoma, and RAd-GFP a vector expressing green fluorescent protein (GFP). The vectors were constructed by the two-plasmid method. These vectors were or are to be complexed with four types of magnetic nanoparticles (MNP) termed, PEI-Mag2 (S9), SO-Mag6-125, PB Mag1-4 and AdenoMag, all possessing a magnetite core and different polymeric coatings. Non complexed vectors were tested at varying multiplicities of infection (MOI) in different cell lines (HEK 293, B92, N2a and A549 cells) and showed pancellular expression of the corresponding reporter gene. In the rat brain, the vectors showed appropriate levels of expression in the hypothalamus, substantia nigra and the ependymal cell layer. Subsequently, MNP-RAd complexes were generated and tested on the above cell lines under the influence of an external magnetic field. Magnetic field-assisted reporter gene delivery (magnetotransduction) led to a MNP-dose dependent enhancement of gene expression. We conclude that our MNP-RAd complexes are suitable for magnetotransduction in vitro. These magnetic adenovectors are to be used in in vivo magnetotransduction studies in the rat brain.. Magnetofection is a methodology developed in the early 2000?fs by the group led by C. Plank in Munich. It is based on the association of MNP with nonviral or viral vectors in order to optimize gene delivery in the presence of a magnetic field. In order to implement magnetofection in the brain we have constructed two adenoviral vectors, RAd-DsRed2 which expresses DsRed2, a red fluorescent protein from Discosoma, and RAd-GFP a vector expressing green fluorescent protein (GFP). The vectors were constructed by the two-plasmid method. These vectors were or are to be complexed with four types of magnetic nanoparticles (MNP) termed, PEI-Mag2 (S9), SO-Mag6-125, PB Mag1-4 and AdenoMag, all possessing a magnetite core and different polymeric coatings. Non complexed vectors were tested at varying multiplicities of infection (MOI) in different cell lines (HEK 293, B92, N2a and A549 cells) and showed pancellular expression of the corresponding reporter gene. In the rat brain, the vectors showed appropriate levels of expression in the hypothalamus, substantia nigra and the ependymal cell layer. Subsequently, MNP-RAd complexes were generated and tested on the above cell lines under the influence of an external magnetic field. Magnetic field-assisted reporter gene delivery (magnetotransduction) led to a MNP-dose dependent enhancement of gene expression. We conclude that our MNP-RAd complexes are suitable for magnetotransduction in vitro. These magnetic adenovectors are to be used in in vivo magnetotransduction studies in the rat brain.in vitro. These magnetic adenovectors are to be used in in vivo magnetotransduction studies in the rat brain.in vivo magnetotransduction studies in the rat brain.