The transient expression of genes encoded by minicircles enables the overexpression and/or prolonged expression of specific signaling molecules to modulate cell survival, proliferation or differentiation [1], foreseeing the maximization of the therapeutic potential of stem cells for applications in Cellular and Gene Therapy, as well as Tissue Engineering. Minicircles are safe and effective because are small molecules devoid of specific DNA sequences that may trigger gene silencing and the attack of the immune system.
At SCERG, gene induced modification of human mesenchymal stem/stromal cells (MSC) [2,3] is pursued towards the maximization of the expansion and differentiation potential of these cells, but also to improve their intrinsic therapeutic features. In this context, an ex vivo gene therapy strategy is envisaged to engineer MSC from different human sources [3] with minicircles in order to secrete modulating proteins and exosomes that will alter the behavior of targeting cells.
Minicircle non-viral vectors are rationally engineered [4] to increase ex vivo MSC transfection, extend transgene expression and facilitate purification [5] (collaboration with BERG). Minicircle gene delivery by microporation [1] and by nano- and microparticles are being developed to obtain more efficient gene delivery strategies [6,7]. Additionally, the composition of the cell secretome that contains several types of signaling molecules, including microRNAs, may be modified by minicircle-encoded genes and used to alter the response of target cells.
Selected publications:
[1] Madeira, C., Rodrigues, C.A.V., Reis, M.S.C., Ferreira, F.C.G., Correia, R.E.S.M., Diogo, M.M., Cabral, J.M.S. Non-viral gene delivery to neural stem cells with minicircles by microporation. Biomacromolecules 14(5):1379-1387 (2013).
[2] Madeira, C., Ribeiro, S.C., Pinheiro, I.S.M., Martins, S.A.M., Andrade, P.Z., da Silva, C.L., Cabral, J.M.S. Gene delivery to human bone marrow mesenchymal stem cells by microporation. J Biotechnol 151(1):130-136 (2011).
[3] Boura, J.S., dos Santos, F., Gimble, J.M., Cardoso, C.M., Madeira, C., Cabral, J.M., da Silva, C.L. Direct head-to-head comparison of cationic liposome-mediated gene delivery to mesenchymal stem/stromal cells of different human sources: a comprehensive study. Hum Gene Ther Methods 24(1):38-48 (2013).
[4] Šimčíková, M., Alves, C.P.A., Brito, L., Prather, K.L.J., Prazeres, D.M.F., Monteiro, G.A. Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase. Appl Micro Biotechnol 100:6725-6737 (2016).
[5] Alves, C.P.A., Šimčíková, M., Prather, K.L.J., Monteiro, G.A., Prazeres, D.M.F. Development of a nicking endonuclease-assisted method for the purification of minicircles J Chromatog A 1443:136-144 (2016).
[6] Matos, J. C., Soares, A.R., Domingues, I., Monteiro, G.A., Gonçalves, M.C. ORMOPLEXEs for gene therapy: in vitro and in vivo assays. Mater Sci Eng C 63:546-553 (2016).
[7] Magalhães, S., Duarte, S., Monteiro, G.A., Fernandes, F. Quantitative evaluation of DNA dissociation from liposome carriers and DNA escape from endosomes during lipid mediated gene delivery. Hum Gene Ther Method 25:303-313 (2014).