Maria C. Pedroso de Lima

NATO ASI Series

The purpose of this presentation is to describe procedures of analysis of final extents and kinetics of virus fusion with target membranes. The presentation of results will focus on deductions from studies of final extents of fusion.

Cationic lipid-DNA complexes in gene delivery : from biophysics to biological applications

Great expectations from the application of gene therapy approaches to human disease have been impaired by the unsatisfactory clinical progress observed. Among others, the use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of this promising therapeutic strategy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. This review focuses on the various parameters that are considered to be crucial to optimize the use of cationic lipid-DNA complexes for gene therapy purposes. Particular emphasis is devoted to the analysis of the different stages involved in the transfection process, from the biophysical aspects underlying the formation of the complexes to the different biological barriers that need to be surpassed for gene expression to occur.

Sterically Stabilized pH-sensitive Liposomes INTRACELLULAR DELIVERY OF AQUEOUS CONTENTS AND PROLONGED CIRCULATION IN VIVO

Liposomes that destabilize at mildly acidic pH are efficient tools for delivering water-soluble drugs into the cell cytoplasm. However, their use in vivo is limited because of their rapid uptake from circulation by the reticuloendothelial system. Lipid-anchored polyethylene glycol (PEG-PE) prolongs the circulation time of liposomes by steric stabilization. We have found that addition of PEG-PE to the membrane of pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) confers steric stability to these vesicles. This modification significantly decreases the pH-dependent release of a charged water-soluble fluorophore, calcein, from liposomes suspended in buffer or cell culture medium. However, the ability of such liposomes to release calcein intracellularly, measured by a novel flow cytometry technique involving dual fluorescence labeling, remains unaltered. As expected, the release of calcein from liposomes endocytosed by cells is inhibited upon pretreatment of the cells with NH4Cl, an inhibitor of endosome acidification. The unique properties of these liposomes were also demonstrated in vivo. The distribution kinetics of 111In-containing CHEMS/DOPE/PEG-PE liposomes injected intravenously into rats has pharmacokinetic parameters similar to control, non-pH-sensitive, sterically stabilized CHEMS/distearoylphosphatidylcholine/PEG-PE liposomes. In contrast, regular pH-sensitive liposomes lacking the PEG-PE component are cleared rapidly. Sterically stabilized pH-sensitive liposomes may therefore be useful for the intracellular delivery in vivo of highly negatively charged molecules such as genes, antisense oligonucleotides, and ribozymes for the treatment of various diseases.

Cationic liposomes for gene delivery

Cationic liposome–DNA complexes (lipoplexes) constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. This review will focus on various parameters governing lipoplex biological activity, from their mode of formation to invivo behaviour. Particular emphasis is given to the mechanism of interaction of lipoplexes with cells, in an attempt to dissect the different barriers that need to be surpassed for efficient gene expression to occur. Aspects related to new trends in the formulation of lipid-based gene delivery systems aiming at overcoming some of their limitations will be covered. Finally, examples illustrating the potential of ca-tionic liposomes in clinical applications will be provided.

Cell-Penetrating Peptides - Mechanisms of Cellular Uptake and Generation of Delivery Systems

The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S413-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides). In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S413-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid delivery.

miR‐155 modulates microglia‐mediated immune response by down‐regulating SOCS‐1 and promoting cytokine and nitric oxide production

Innate immunity constitutes the first line of defence against both external and endogenous threats in the brain, and microglia cells are considered key mediators of this process. Recent studies have shown that microRNAs (miRNAs) may play a determinant role in the regulation of gene expression during innate immune responses. The major goal of this work was to investigate the contribution of a specific miRNA – miR‐155 – to the modulation of the microglia‐mediated immune response. For this purpose, in vitro studies were performed in N9 microglia cells to evaluate changes in the levels of this miRNA following microglia activation. A strong up‐regulation of miR‐155 expression was observed following microglia exposure to lipopolysaccharide, which was consistent with a decrease in the levels of the suppressor of cytokine signalling 1 (SOCS‐1) protein, a key inhibitor of the inflammatory process and a predicted target of miR‐155. The miR‐155 knockdown by anti‐miRNA oligonucleotides up‐regulated SOCS‐1 mRNA and protein levels and significantly decreased the production of nitric oxide and the expression of inflammatory cytokines and inducible nitric oxide synthase. Finally, treatment of neuronal primary cultures with conditioned medium obtained from microglia cells, in which miR‐155 was inhibited before cell activation, decreased inflammatory‐mediated neuronal cell death. Overall, our results show that miR‐155 has a pro‐inflammatory role in microglia and is necessary for the progression of the immune response through the modulation of SOCS‐1, suggesting that, in a chronic inflammatory context, miR‐155 inhibition can have a neuroprotective effect.

Lipid-based nanoparticles for siRNA delivery in cancer therapy: paradigms and challenges.

RNA interference (RNAi) is a specific gene-silencing mechanism that can be mediated by the delivery of chemical synthesized small-interfering RNA (siRNA). RNAi might constitute a novel therapeutic approach for cancer treatment because researchers can easily design siRNA molecules to inhibit, specifically and potently, the expression of any protein involved in tumor initiation and progression. Despite all the potential of siRNA as a novel class of drugs, the limited cellular uptake, low biological stability, and unfavorable pharmacokinetics of siRNAs have limited their application in the clinic. Indeed, blood nucleases easily degrade naked siRNAs, and the kidneys rapidly eliminate these molecules. Furthermore, at the level of target cells, the negative charge and hydrophilicity of siRNAs strongly impair their cellular internalization. Therefore, the translation of siRNA to the clinical setting is highly dependent on the development of an appropriate delivery system, able to ameliorate siRNA pharmacokinetic and biodistribution properties. In this regard, major advances have been achieved with lipid-based nanocarriers sterically stabilized by poly(ethylene glycol) (PEG), such as the stabilized nucleic acid lipid particles (SNALP). However, PEG has not solved all the major problems associated with siRNA delivery. In this Account, the major problems associated with PEGylated lipid-based nanoparticles, and the different strategies to overcome them are discussed. Although PEG has revolutionized the field of nanocarriers, cumulative experience has revealed that upon repeated administration, PEGylated liposomes lose their ability to circulate over long periods in the bloodstream, a phenomenon known as accelerated blood clearance. In addition, PEGylation impairs the internalization of the siRNA into the target cell and its subsequent escape from the endocytic pathway, which reduces biological activity. An interesting approach to overcome such limitations relies on the design of novel exchangeable PEG-derivatized lipids. After systemic administration, these lipids can be released from the nanoparticle surface. Moreover, the design and synthesis of novel cationic lipids that are more fusogenic and the use of internalizing targeting ligands have contributed to the emergence of novel lipid-based nanoparticles with remarkable transfection efficiency.

Suicide gene therapy in cancer: Where do we stand now?

Suicide gene therapy is based on the introduction into tumor cells of a viral or a bacterial gene, which allows the conversion of a non-toxic compound into a lethal drug. Although suicide gene therapy has been successfully used in a large number of in vitro and in vivo studies, its application to cancer patients has not reached the desirable clinical significance. However, recent reports on pre-clinical cancer models demonstrate the huge potential of this strategy when used in combination with new therapeutic approaches. In this review, we summarize the different suicide gene systems and gene delivery vectors addressed to cancer, with particular emphasis on recently developed systems and associated bystander effects. In addition, we review the different strategies that have been used in combination with suicide gene therapy and provide some insights into the future directions of this approach, particularly towards cancer stem cell eradication.

On the mechanisms of internalization and intracellular delivery mediated by pH-sensitive liposomes

We investigated the molecular mechanisms by which pH-sensitive liposomes surpass the cytoplasmic and endosomal membranes to deliver their aqueous contents into the cytoplasm. Various liposome formulations were evaluated for their efficacy to mediate intracellular delivery of encapsulated material, including a novel sterically stabilized pH-sensitive formulation ((DOPE:CHEMS:DSPE-PEG(2000) (6:4:0.3)) that was previously developed in our laboratories. In an attempt to fully characterize the nature of liposome-cell interactions different approaches based on a dual-labeling fluorescence assay were used. Our results indicate that the efficacy of interaction of pH-sensitive liposomes, both plain and sterically stabilized, with cells is strongly determined by the inclusion of DOPE in their composition, independently of the type of the amphiphilic stabilizer used. In fact, DOPE-containing liposomes shown to be non-pH sensitive by biophysical assays, mediated cytoplasmic delivery of their contents as efficiently as well known pH-sensitive formulations (e.g. DOPE:CHEMS). However, among the different formulations studied, DOPE:CHEMS liposomes were those exhibiting the highest extent of cell association. Moreover, our results with cells pretreated with metabolic inhibitors or lysosomotropic agents clearly indicate that DOPE-containing liposomes are internalized essentially by endocytosis and that acidification of the endosomes is not the only mechanism involved in the destabilization of the liposomes inside the cell.

Allele-Specific RNA Silencing of Mutant Ataxin-3 Mediates Neuroprotection in a Rat Model of Machado-Joseph Disease

Recent studies have demonstrated that RNAi is a promising approach for treating autosomal dominant disorders. However, discrimination between wild-type and mutant transcripts is essential, to preserve wild-type expression and function. A single nucleotide polymorphism (SNP) is present in more than 70% of patients with Machado-Joseph disease (MJD). We investigated whether this SNP could be used to inactivate mutant ataxin-3 selectively. Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo. The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD. These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.