Annual Report of Activities CNC 2008 - Center for Neuroscience and ...

Vectors and Gene Therapy | Head: Maria da Conceição Pedroso de LimaObjectivesThe Group of Vectors and Gene Therapy isdevoted to the design and development of carriers,including viral and non‐viral vectors, for nucleicacid and drug delivery aiming at their applicationin gene therapy and gene silencing approaches.Regarding the development of non‐viral vectors,the specific aims include the generation of novellipid‐based nanosystems for efficient intracellulardelivery of drugs and nucleic acids (plasmid DNA,antisense oligonucleotides or siRNAs) andevaluation of their potential in therapeuticapproaches for two major diseases: cancer andneurodegenerative disorders. For this purpose, ourresearch has been focused on the selection ofappropriate lipids, ligands and cell‐penetratingpeptides, and on the development of technology togenerate nanosystems with adequate features for invivo use, allowing targeting to specific cells ortissues and enhanced intracellular nucleic aciddelivery. In parallel, mechanistic studies on theinteraction of the developed systems with targetcells, including cell internalization and intracellulartrafficking, have also been addressed aiming attheir optimization for specific therapeuticapplications. Evaluation of the therapeutic activitymediated by the developed systems has beenperformed in several in vitro and in vivo modelsfor cancer and neurodegenerative disorders, andalso constitutes an important goal of this Group.Regarding viral vectors, the group aims atdeveloping and using viral vectors as atechnological platform to generate genetic modelsof neurodegenerative diseases, such as Machado‐Joseph disease, and to develop new moleculartherapeutic strategies involving gene transfer orsilencing of mutant genes by expression of shorthairpin RNAs.Main AchievementsRegarding the development of improved non‐viralgene delivery vectors, we demonstrated thecapacity of the S413‐PV cell‐penetrating peptide,either per se or in association with cationicliposomes, to very efficiently mediate theintracellular delivery of plasmid DNA.Regarding the evaluation of the developednanosystems in anticancer strategies, we haveshown that combination of antineoplastic agentswith suicide gene therapy mediated by albuminassociatedlipoplexes results in a remarkablesynergistic antitumor effect, highlighting thepotential of this approach for future applications inantitumoral therapy.We have also developed a novel lipid‐basednanosystem that has the potential to target themicroenvironment of human breast tumors at twodifferent levels: tumor cells and endothelial cells oftumor blood vessels. Such features led to adramatic improvement of cytotoxicity ofencapsulated small molecular weight drug, ascompared to the non‐targeted formulation. Thetargeting ability of the developed nanosystem wasconfirmed in tumor cells harvested from tumors ofpatients submitted to mastectomy.Concerning the potential of the developednanosystems in gene silencing approaches forneurodegenerative disorders, we achievedsignificant downregulation of gene expressionupon neuronal siRNA delivery mediated bytransferrin‐associated lipoplexes, both in vitro andin vivo. Moreover, promising results were obtainedregarding the application of these systems tomediate downregulation of specific pro‐apoptotictargets, which may prove useful in therapeuticapproaches to neuronal protection and repair.Using lentiviral vectors (LV) to transduce the ratbrain we overexpressed polyglutamine‐expandedataxin‐3 in this way replicating Machado‐Josephdisease neuropathology. LVs also allowed the firstdemonstration of in vivo allele‐specific genesilencing. More recently we did not observetoxicity upon endogenous wild‐type ataxin‐3silencing and showed that LV‐mediated non‐allelespecificsilencing of ataxin‐3 is effective and welltolerated in vivo.38